Method, array, and kit for detecting activated transcription factors

ABSTRACT

Methods, arrays and kits are provided for rapidly and efficiently identifying and quantifying multiple different activated transcription factors in a biological sample at the same time. In one embodiment, the method includes the step of mixing a library of different transcription factor probes with a sample containing activated transcription factors. The transcription factor probes that have bound to the activated transcription factors may be isolated from the complexes formed between the probes and the activated transcription factors. The bound probes can be identified, for example, by using an array of hybridization probes.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of U.S. application Ser. No.09/877,243, filed on Jun. 8, 2001, and is hereby incorporated herein byreference.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for detecting activatedtranscription factors in a cell sample. More specifically, the inventionrelates to methods for detecting multiple different activatedtranscription factors in a cell sample at the same time and uses arisingthere from.

DESCRIPTION OF RELATED ART

[0003] All living organisms use nucleic acids (DNA and RNA) to encodethe genes which make up the genome for that organism. Each gene encodesa protein that may be produced by the organism through expression of thegene.

[0004] It is important to note that the mere presence of a gene in acell does not communicate the functionality of that gene to the cell.Rather, it is only when the gene is expressed and a protein is producedthat the functionality of the gene encoding the protein is conveyed.

[0005] The systems that regulate gene expression respond to a widevariety of developmental and environmental stimuli, thus allowing eachcell type to express a unique and characteristic subset of its genes,and to adjust the dosage of particular gene products as needed. Theimportance of dosage control is underscored by the fact that targeteddisruption of key regulatory molecules in mice often results in drasticphenotypic abnormalities [Johnson, R. S., et al., Cell, 71:577-586(1992)], just as inherited or acquired defects in the function ofgenetic regulatory mechanisms contribute broadly to human disease.

[0006] The importance of controlled gene expression in human disease andthe information available to date relating to the mechanisms of generegulation have fueled efforts aimed at discovering ways of overridingendogenous regulatory controls or of creating new signaling circuitry incells [Belshaw, P. J., et al., Proc. Natl. Acad. Sci. USA, 93:4604-4607(1996); Ho, S. H., et al., Nature (London), 382:822-826 (1996); Rivera,V. M., et al., Nat. Med., 2:1028-1032; Spencer, D. M., et al., Science,262:1019-1024 (1993)].

[0007] Critical to this research are effective tools for monitoring geneexpression. It is therefore of interest to be able to rapidly andaccurately determine the relative expression of different genes indifferent cells, tissues and organisms, over time, and under variousconditions, treatments and regimes. As will be described herein ingreater detail, there are a great many applications that arise frombeing able to effectively monitor which genes are being expressed by agiven cell at a given time.

[0008] Standard molecular biology techniques have been used to analyzethe expression of genes in a cell by measuring DNA. These techniquesinclude PCR, northern blot analysis, or other types of DNA probeanalysis such as in situ hybridization. Each of these methods allows oneto analyze the transcription of only known genes and/or small numbers ofgenes at a time. Nucl. Acids Res. 19, 7097-7104 (1991); Nucl. Acids Res.18, 4833-4842 (1990); Nucl. Acids Res. 18, 2789-2792 (1989); European J.Neuroscience 2, 1063-1073 (1990); Analytical Biochem. 187, 364-373(1990); Genet. Annal Techn. Appl. 7, 64-70 (1990); GATA 8(4), 129-133(1991); Proc. Natl. Acad. Sci. USA 85, 1696-1700 (1988); Nucl. AcidsRes. 19, 1954 (1991); Proc. Natl. Acad. Sci. USA 88, 1943-1947 (1991);Nucl. Acids Res. 19, 6123-6127 (1991); Proc. Natl. Acad. Sci. USA 85,5738-5742 (1988); Nucl. Acids Res. 16, 10937 (1988).

[0009] Gene expression has also been monitored by measuring levels ofmRNA. Since proteins are transcribed from mRNA, it is possible to detecttranscription by measuring the amount of mRNA present. One commonmethod, called “hybridization subtraction”, allows one to look forchanges in gene expression by detecting changes in mRNA expression.Nucl. Acids Res. 19, 7097-7104 (1991); Nucl. Acids Res. 18, 4833-4842(1990); Nucl. Acids Res. 18, 2789-2792 (1989); European J. Neuroscience2, 1063-1073 (1990); Analytical Biochem. 187, 364-373 (1990); Genet.Annal Techn. Appl. 7, 64-70 (1990); GATA 8(4), 129-133 (1991); Proc.Natl. Acad. Sci. USA 85, 1696-1700 (1988); Nucl. Acids Res. 19, 1954(1991); Proc. Natl. Acad. Sci. USA 88, 1943-1947 (1991); Nucl. AcidsRes. 19, 6123-6127 (1991); Proc. Natl. Acad. Sci. USA 85, 5738-5742(1988); Nucl. Acids Res. 16, 10937 (1988).

[0010] Gene expression has also been monitored by measuring levels ofgene product, (i.e., the expressed protein), in a cell, tissue, organsystem, or even organism. Measurement of gene expression by measuringthe protein gene product may be performed using antibodies known to bindto a particular protein to be detected. A difficulty arises in needingto generate antibodies to each protein to be detected. Measurement ofgene expression via protein detection may also be performed using2-dimensional gel electrophoresis, wherein proteins can be, inprinciple, identified and quantified as individual bands, and ultimatelyreduced to a discrete signal. In order to positively analyze each band,each band must be excised from the membrane and subjected to proteinsequence analysis using Edman degradation. Unfortunately, it tends to bedifficult to isolate a sufficient amount of protein to obtain a reliablesequence. In addition, many of the bands contain more than one discreteprotein.

[0011] A further difficulty associated with quantifying gene expressionby measuring an amount of protein gene product in a cell is that proteinexpression is an indirect measure of gene expression. It is impossibleto know from a protein present in a cell when that protein was expressedby the cell. As a result, it is hard to determine whether proteinexpression changes over time due to cells being exposed to differentstimuli.

[0012] Gene expression has also been monitored by measuring the amountof particular activated transcription factors present in a cell.Transcription in a cell is controlled by proteins, referred to herein as“activated transcription factors” which bind to DNA at sites outside thecore promoter for the gene and activate transcription. Since activatedtranscription factors activate transcription, detection of theirpresence is useful for measuring gene expression. Transcriptionalactivators are found in prokaryotes, viruses, and eukaryotes, includingfungi, plants, and animals, including mammals, providing a wide range oftherapeutic targets.

[0013] The regulatory mechanisms controlling the transcription ofprotein-coding genes by RNA polymerase II have been extensively studied.RNA polymerase II and its host of associated proteins are recruited tothe core promoter through non-covalent contacts with sequence-specificDNA binding proteins [Tjian, R. and Maniatis, T., Cell, 77:5-8 (1994);Stringer, K. F., Nature (London), 345:783-786 (1990)]. An especiallyprevalent and important subset of such proteins, known as transcriptionfactors, typically bind DNA at sites outside the core promoter andactivate transcription through space contacts with components of thetranscriptional machinery, including chromatin remodeling proteins[Tjian, R. and Maniatis, T., Cell, 77:5-8 (1994); Stringer, K. F.,Nature (London), 345:783-786 (1990); Bannister, A. J. and Kouzarides,T., Nature, 384:641-643 (1996); Mizzen, C. A., et al., Cell,87:1261-1270 (1996)]. The DNA-binding and activation functions oftranscription factors generally reside on separate domains whoseoperation is portable to heterologous fusion proteins [Sadowski, I., etal., Nature, 335:563-564 (1988)]. Though it is believed that activationdomains are physically associated with a DNA-binding domain to attainproper function, the linkage between the two need not be covalent[Belshaw, P. J., et al., Proc. Natl. Acad. Sci. USA, 93:4604-4607(1996); Ho, S. H., et al., Nature (London), 382:822-826 (1996)]. In manyinstances, the activation domain does not appear to contact thetranscriptional machinery directly, but rather through the intermediacyof adapter proteins known as coactivators [Silverman, N., et al., Proc.Natl. Acad. Sci. USA, 91:11005-11008 ((1994); Arany, Z., et al., Nature(London), 374:81-84 (1995)].

[0014] One of the difficulties associated with measuring gene expressionby measuring transcription factors is that one must measure the subsetof transcription factors which are “activated.” Certainpost-transcriptional modifications occur which render transcriptionfactors “active” in the sense that they are capable of binding to DNA.It is thus necessary to distinguish between activated and non-activatedtranscription factors so that the “activated transcription factors” canbe selectively measured.

[0015] Several different methods have been developed for detectingactivated transcription factors. One method involves using antibodiesselective for activated transcription factors over inactive forms of thetranscription factor. This method is impractical for detecting multipledifferent activated transcription factors due to difficulties associatedwith developing numerous different antibodies having the requisite bindspecificities.

[0016] Another method for detecting activated transcription factorsinvolves measuring DNA-transcription factor complexes through a gelshift assay. [Ausebel, F. M. et al eds (1993) Current Protocols inMolecular Biology Vol.2 Greene Publishing Associates, Inc. and JohnWiley and Sons, Inc., New York]. According to this method, a samplecontaining an activated transcription factor is contacted with a DNAprobe that comprises a recognition sequence for the transcriptionfactor. A complex between the activated transcription factor and the DNAprobe is formed. The DNA-protein complex is detected by a gel-shiftassay. Since individual gel shift assays must be performed for eachactivated transcription factor-DNA complex, this method is currentlyimpractical for measuring multiple different activated transcriptionfactors at the same time.

[0017] U.S. Pat. Nos. 6,066,452 and 5,861,246 describe methods fordetermining DNA binding sites for DNA-binding proteins. The DNA bindingsites may then be used as probes to isolate DNA-binding proteins.Similarly, PCT Publication No. WO 00/04196 describes methods foridentifying cis acting nucleic acid elements as well as methods forisolating nucleic acid binding factors.

SUMMARY OF THE INVENTION

[0018] The present invention relates to methods and kits for isolatingDNA probes that bind to activated transcription factors.

[0019] In one embodiment, a method is provided which comprises:contacting a biological sample with a library of double stranded DNAprobes under conditions where DNA probe-transcription factor complexesare formed between the DNA probes and activated transcription factorspresent in the biological sample; separating DNA probe-transcriptionfactor complexes from non-complexed DNA probes in the library using anagarose gel separation; excising a portion of the agarose gel comprisingthe separated DNA probe-transcription factor complexes; and isolatingthe DNA probes from the excised portion of the agarose gel.

[0020] In another embodiment, a method is provided which comprises:contacting a biological sample with a library of double stranded DNAprobes under conditions where DNA probe-transcription factor complexesare formed between the DNA probes and activated transcription factorspresent in the biological sample; separating DNA probe-transcriptionfactor complexes from non-complexed DNA probes in the library using anagarose gel separation; excising a portion of the agarose gel comprisingthe separated DNA probe-transcription factor complexes; isolating theDNA probes from the excised portion of the agarose gel; and identifyingwhich of the DNA probes in the library are isolated.

[0021] In another embodiment, a kit is provided which comprises: alibrary of double stranded DNA probes, each probe comprising arecognition sequence to which an activated transcription factor iscapable of binding and forming a DNA probe-transcription factor complex,the DNA probes in the library capable of forming DNA probe-transcriptionfactor complexes with multiple different activated transcriptionfactors; and instructions for separating DNA probe-transcription factorcomplexes from non-complexed DNA probes in the library by agarose gelseparation.

[0022] Kits are also provided for DNA probe libraries for detectingactivated transcription factors.

[0023] In one embodiment, the kit comprises: first and second librariesof double stranded DNA probes, each probe in the first and secondlibraries comprising a recognition sequence to which an activatedtranscription factor is capable of binding and forming a DNAprobe-transcription factor complex, the DNA probes in the librarycapable of forming DNA probe-transcription factor complexes withmultiple different activated transcription factors; wherein the probesof the first library further comprise a first detectable marker and theprobes of the second library further comprise a second detectable markerthat is different than the first detectable marker.

[0024] Methods, arrays and kits are also provided for detectingactivated transcription factors using a hybridization array.

[0025] In one embodiment, a method is provided which comprises: taking alibrary of double stranded transcription factor probes, thetranscription factor probes each comprising a recognition sequencecapable of binding to an activated transcription factor, the recognitionsequence varying within the library for binding to different activatedtranscription factors; contacting a biological sample with the libraryof double stranded DNA probes under conditions where DNAprobe-transcription factor complexes are formed between the DNA probesand activated transcription factors present in the biological sample;isolating the transcription factor probes from the transcription factorprobe-transcription factor complexes formed; and identifying whichtranscription factor probes in the library formed complexes by taking anarray of immobilized hybridization probes capable of hybridizing to atleast one of the strands of the different double stranded transcriptionfactor probes in the library and contacting the isolated transcriptionfactor probes with the array under conditions suitable for hybridizationof the strands of the different double stranded transcription factorprobes to the hybridization probes in the array.

[0026] In another embodiment, a hybridization array is provided for usein identifying which of a plurality of different activated transcriptionfactors are present in a biological sample by immobilizing transcriptionfactor probes that form transcription factor probe-transcription factorcomplexes with different activated transcription factors, the arraycomprising: a substrate; and a plurality of hybridization probesimmobilized on a surface of the substrate such that differenthybridization probes are positioned in different defined regions on thesurface, the different hybridization probes comprising a differenttranscription factor probe binding region capable of immobilizing adifferent transcription factor probe to the array, the transcriptionfactor probe binding region comprising at least two copies of acomplement to a portion of a recognition sequence comprised on thetranscription factor probe. The hybridization array may optionallyfurther comprise an internal standard. For example, the array mayfurther comprise biotinylated DNA which is employed as an internalstandard.

[0027] In another embodiment, a kit is provided for use in identifyingwhich of a plurality of different activated transcription factors arepresent in a biological sample by isolating and immobilizingtranscription factor probes that form transcription factorprobe-transcription factor complexes with different activatedtranscription factors, the kit comprising: a hybridization arraycomprising a substrate, and a plurality of hybridization probesimmobilized on a surface of the substrate such that differenthybridization probes are positioned in different defined regions on thesurface, the different hybridization probes comprising a differenttranscription factor probe binding region capable of immobilizing adifferent transcription factor probe to the array, the transcriptionfactor probe binding region comprising at least two copies of acomplement to a portion of a recognition sequence comprised on thetranscription factor probe; and instructions for separating DNAprobe-transcription factor complexes from non-complexed DNA probes inthe library by agarose gel separation.

[0028] Methods for characterizing cell types based on which activatedtranscription factors are present in a sample are also provided.

[0029] In one embodiment, a method is provided which comprises: taking alibrary of double stranded transcription factor probes, thetranscription factor probes each comprising a recognition sequencecapable of binding to an activated transcription factor, the recognitionsequence varying within the library for binding to different activatedtranscription factors native to different cell types; contacting abiological sample with the library of double stranded DNA probes underconditions where DNA probe-transcription factor complexes are formedbetween the DNA probes and activated transcription factors present inthe biological sample; isolating the transcription factor probes fromthe transcription factor probe-transcription factor complexes formed;identifying which transcription factor probes in the library formedcomplexes by taking an array of immobilized hybridization probes capableof hybridizing to at least one of the strands of the different doublestranded transcription factor probes in the library and contacting theisolated transcription factor probes with the array under conditionssuitable for hybridization of the strands of the different doublestranded transcription factor probes to the hybridization probes in thearray; and identifying a cell type of the biological sample based onwhich transcription factor probes are identified.

[0030] Methods for identifying a disease state based on which activatedtranscription factors are present in a biological sample are alsoprovided.

[0031] In one embodiment, the method comprises taking a library ofdouble stranded transcription factor probes, the transcription factorprobes each comprising a recognition sequence capable of binding to anactivated transcription factor, the recognition sequence varying withinthe library for binding to different activated transcription factorsnative to different cell types; identifying which activatedtranscription factors are present in a nuclear extract of a test sampleof cells by: contacting the nuclear extract of the test sample with thelibrary of double stranded DNA probes under conditions where DNAprobe-transcription factor complexes are formed between the DNA probesand activated transcription factors present in the test sample,isolating the transcription factor probes from the transcription factorprobe-transcription factor complexes formed, and identifying whichtranscription factor probes in the library formed complexes by taking anarray of immobilized hybridization probes capable of hybridizing to atleast one of the strands of the different double stranded transcriptionfactor probes in the library and contacting the isolated transcriptionfactor probes with the array under conditions suitable for hybridizationof the strands of the different double stranded transcription factorprobes to the hybridization probes in the array; identifying whichactivated transcription factors are present in a nuclear extract of acontrol sample of cells by: contacting the nuclear extract of thecontrol sample with the library of double stranded DNA probes underconditions where DNA probe-transcription factor complexes are formedbetween the DNA probes and activated transcription factors present inthe control sample, isolating the transcription factor probes from thetranscription factor probe-transcription factor complexes formed, andidentifying which transcription factor probes in the library formedcomplexes by taking an array of immobilized hybridization probes capableof hybridizing to at least one of the strands of the different doublestranded transcription factor probes in the library and contacting theisolated transcription factor probes with the array under conditionssuitable for hybridization of the strands of the different doublestranded transcription factor probes to the hybridization probes in thearray; and comparing which activated transcription factors are presentin the test sample and the control sample.

[0032] Methods for screening drug candidates for modulating an activatedtranscription factor's activity are also provided.

[0033] In one embodiment, the method comprises: forming a plurality oftest samples by contacting samples of cells with different agents; andfor each test sample, identifying which of a plurality of differentactivated transcription factors are present by: taking a library ofdouble stranded transcription factor probes, the transcription factorprobes each comprising a recognition sequence capable of binding to anactivated transcription factor, the recognition sequence varying withinthe library for binding to different activated transcription factors,contacting the different test sample with the library of double strandedDNA probes under conditions where DNA probe-transcription factorcomplexes are formed between the DNA probes and activated transcriptionfactors present in the test samples, isolating the transcription factorprobes from the transcription factor probe-transcription factorcomplexes formed, and identifying which transcription factor probes inthe library formed complexes by taking an array of immobilizedhybridization probes capable of hybridizing to at least one of thestrands of the different double stranded transcription factor probes inthe library and contacting the isolated transcription factor probes withthe array under conditions suitable for hybridization of the strands ofthe different double stranded transcription factor probes to thehybridization probes in the array; and comparing the activatedtranscription factors present in the different test samples.

[0034] Methods for determining sequence binding requirements for anactivated transcription factor are also provided.

[0035] In one embodiment, the method comprises: contacting a samplecomprising an activated transcription factor with a library of doublestranded DNA probes under conditions where DNA probe-transcriptionfactor complexes are formed between the DNA probes and the activatedtranscription factor; separating DNA probe-transcription factorcomplexes from non-complexed DNA probes in the library; isolating theDNA probes from the excised portion of the agarose gel; and determininga consensus sequence for the DNA probes isolated in order to assess thebinding requirements for the transcription factor.

[0036] In another embodiment, the method comprises contacting a samplecomprising an activated transcription factor with a library of doublestranded DNA probes under conditions where DNA probe-transcriptionfactor complexes are formed between the DNA probes and the activatedtranscription factor; separating DNA probe-transcription factorcomplexes from non-complexed DNA probes in the library using an agarosegel separation; excising a portion of the agarose gel comprising theseparated DNA probe-transcription factor complexes; isolating the DNAprobes from the excised portion of the agarose gel; and determining aconsensus sequence for the DNA probes isolated in order to assess thebinding requirements for the transcription factor.

[0037] In yet another embodiment, the method comprises: contacting asample comprising an activated transcription factor with a library ofdouble stranded DNA probes under conditions where DNAprobe-transcription factor complexes are formed between the DNA probesand the activated transcription factor; separating DNAprobe-transcription factor complexes from non-complexed DNA probes inthe library; isolating the DNA probes from the excised portion of theagarose gel; and quantifying the amount of each of the isolated DNAprobes.

[0038] In yet another embodiment, the method comprises: contacting asample comprising an activated transcription factor with a library ofdouble stranded DNA probes under conditions where DNAprobe-transcription factor complexes are formed between the DNA probesand the activated transcription factor; separating DNAprobe-transcription factor complexes from non-complexed DNA probes inthe library using an agarose gel separation; excising a portion of theagarose gel comprising the separated DNA probe-transcription factorcomplexes; isolating the DNA probes from the excised portion of theagarose gel; and quantifying the amount of each of the isolated DNAprobes.

[0039] Methods are also provided for quantifying expression andactivation of multiple different activated transcription factors.According to one embodiment, the method comprises: contacting abiological sample with a library of double stranded DNA probes fordetecting active forms of multiple different transcription factors underconditions where DNA probe-transcription factor complexes are formedbetween the DNA probes and activated transcription factors present inthe biological sample; isolating DNA probes from the DNAprobe-transcription factor complexes; identifying which of the multipledifferent transcription factors are present in an activated form in thebiological sample based on which DNA probes are isolated; andquantifying expression of the multiple different transcription factorsfrom cDNA for the biological sample.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040]FIG. 1 provides a flow diagram for a method for identifying whichof a plurality of transcription factors are activated in a given sampleof cells.

[0041]FIG. 2 illustrates an array of hybridization probes attached to asolid support where different hybridization probes are attached todiscrete, different regions of the array. A transcription factorexpression signature is shown based on the distribution of wheretranscription factor probes hybridize and their intensity.

[0042]FIG. 3 illustrates an array of hybridization probes attached to asolid support where probes from a first sample with a first color dyeand probes from a second sample with a second color dye are bothcontacted with the array.

[0043]FIG. 4 illustrates a process whereby the minimum DNA sequencebinding requirements for a given transcription factor can be rapidlydetermined.

[0044]FIG. 5 illustrates a variation of the method described in regardto FIG. 4 where an optimal sequence for binding is identified.

[0045]FIG. 6 provides the sequences for the probes used to form thetranscription factor probe library used in the experiments described inSections 12-19 herein.

[0046]FIG. 7 depicts the layout of the array of hybridization probesemployed in the experiments described in Sections 12-19 herein.

[0047]FIG. 8 is an image of the array described in regard to FIG. 7 whenthe transcription factor probe library described in FIG. 6 is contactedwith the array.

[0048]FIG. 9A is an image of an array described in regard to FIG. 7 thatis contacted with transcription factor hybridization probes isolatedfrom transcription factor probe-transcription factor complexes formedwhen Brn3 transcription factor hybridization probes are contacted with anuclear extract of HeLa cells.

[0049]FIG. 9B is an image of an array described in regard to FIG. 7 thatis contacted with transcription factor hybridization probes isolatedfrom transcription factor probe-transcription factor complexes formedwhen c-Myb transcription factor hybridization probes are contacted witha nuclear extract of HeLa cells.

[0050]FIG. 9C is an image of an array described in regard to FIG. 7 thatis contacted with transcription factor hybridization probes isolatedfrom transcription factor probe-transcription factor complexes formedwhen Smad3/4 transcription factor hybridization probes are contactedwith a nuclear extract of HeLa cells.

[0051]FIG. 9D is an image of an array described in regard to FIG. 7 thatis contacted with transcription factor hybridization probes isolatedfrom transcription factor probe-transcription factor complexes formedwhen Brn3, c-Myb, and Smad3/4 transcription factor hybridization probesare contacted with a nuclear extract of HeLa cells.

[0052]FIG. 10A is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a control sample that does notcontain any transcription factors.

[0053]FIG. 10B is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a nuclear extract of HeLa cells.

[0054]FIG. 11A is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a nuclear extract of HeLa cells.

[0055]FIG. 11B is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a nuclear extract of PMA-treatedHeLa cells.

[0056]FIG. 12A provides a table of the signal intensities of regions ofthe array shown in FIG. 11A.

[0057]FIG. 12B provides a table of the signal intensities of regions ofthe array shown in FIG. 11B.

[0058]FIG. 12C provides a table with the ratios between the intensitiesof the regions of the arrays shown in FIGS. 12A and 12B.

[0059]FIG. 13 provides an image of a gel shift analysis of Est and NF-E1performed on HeLa and PMA-treated HeLa cells.

[0060]FIG. 14A is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a nuclear extract of A431 cells.

[0061]FIG. 14B is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a nuclear extract of PMA-treatedA431 cells.

[0062]FIG. 15 provides an image of a gel shift analysis of Ets, NF-E1,and NF-kB performed on A431 and PMA-treated A431 cells.

[0063]FIG. 16A is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a nuclear extract of Jurkatcells.

[0064]FIG. 16B is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a nuclear extract of PMA-treatedJurkat cells.

[0065]FIG. 17A is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a nuclear extract of HeLa cells.

[0066]FIG. 17B is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a nuclear extract of A431 cells.

[0067]FIG. 17C is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a nuclear extract of Jurkatcells.

[0068]FIG. 17D is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a nuclear extract of K-562 cells.

[0069]FIG. 17E is an image of an array described in regard to FIG. 7that is contacted with transcription factor hybridization probesisolated from transcription factor probe-transcription factor complexesformed when the entire library of transcription factor probes describedin regard to FIG. 6 are contacted with a nuclear extract of Y79 cells.

[0070]FIG. 18A is an image of a polyacrylamide gel from a gel shiftanalysis for multiple different transcription factors.

[0071]FIG. 18B is an image of an agarose gel from a gel shift analysisfor multiple different transcription factors.

DETAILED DESCRIPTION OF THE INVENTION

[0072] The present invention relates to rapid and efficient methods foridentifying multiple different activated transcription factors in abiological sample at the same time. The methods of the present inventionalso provide for the quantification of the multiple different activatedtranscription factors. As will be described herein in greater detail,there are a great many applications that arise from being able toeffectively monitor which genes are being expressed at a given time.With the assistance of the methods of the present invention, it is thuspossible to rapidly and effectively monitor the levels of expression ofmultiple different genes at the same time.

[0073] The present invention also relates to various compositions, kits,and devices for use in conjunction with the various methods of thepresent invention.

[0074]FIG. 1 provides a general overview of how the present inventiondetects multiple different activated transcription factors and thusallows the expression of multiple different genes to be simultaneouslymonitored.

[0075] As illustrated, a biological sample is contacted with a libraryof probes. The biological sample is typically derived from a sample ofcells and is preferably a nuclear extract of the cell sample. The samplecontains an unknown mixture of activated transcription factors. Whichactivated transcription factors are present in the sample serves toindicate which genes are currently being expressed.

[0076] Information about the DNA binding specificity of transcriptionfactors which one wishes to identify is used to design a library oftranscription factor probes. In this regard, it is noted that thepresent invention relates to the detection, monitoring and optionallyquantification of known transcription factors using a library oftranscription factor probes whose sequences are known. The probes in thelibrary are preferably double stranded. One of the strands is preferablybiotin labeled at the 5′ end to facilitate detection.

[0077] Each transcription factor probe in the library comprises a DNAsequence that is capable of binding to an activated transcriptionfactor, referred to herein as the probe's recognition sequence. At leastthe recognition sequence varies within the library of probes such thatthe probes are capable of binding to a plurality of different activatedtranscription factors. Due to the high level binding specificity of thetranscription factors, each transcription factor probe has bindingspecificity for a single transcription factor or family of transcriptionfactors.

[0078] Because the present invention is used to identify knowntranscription factors, it is practical to use longer recognitionsequences in the probes in the library as compared to what would bepractical if a random library were used. As a result, at least 1%, 2%,3%, 5%, 10%, 20%, 30%, 50% or more of the probes in the library may haverecognition sequences greater than 35, 40, 45 or more base pairs inlength. By using longer recognition sequences, the probes have greaterbinding specificity. In addition, the probes have greater bindingefficiency to the transcription factors which improves the yield ofprobe-transcription factor complexes isolated. As a result, the methodof the present invention provides a high level of sensitivity forisolating probe-transcription factor complexes, as described furtherherein, in combination with a high signal to noise ratio.

[0079] As a result of contacting the sample with the library oftranscription factor probes, complexes are formed between activatedtranscription factors present in the sample and transcription factorprobes in the library which have sequences that match the sequencespecificity of the DNA-binding domains of the activated transcriptionfactors. By isolating the transcription factor probe-activatedtranscription factor complexes, those probes from the library which bindto transcription factors in the sample are isolated.

[0080] The isolated transcription factor probes are then identified.Each probe is specific for a different transcription factor. Since onlythose probes from the library which form a complex with an activatedtranscription factor will be isolated, identification of which probesare isolated serves to identify which activated transcription factorsare present. Since the presence of an activated transcription factorevidences gene expression, the above described method can be used todetermine which genes were being expressed at the time the sample wastaken based on which activated transcription factors are present.

[0081] The design, operation and applications for the present inventionwill now be described in greater detail.

[0082] 1. Libraries of Transcription Factor Probes

[0083] Libraries of transcription factor probes are provided that may beused to detect activated transcription factors according to the presentinvention. A given library comprises a plurality of double stranded DNAprobes where the DNA sequences of the probes vary within the library.The DNA sequences employed in the probes of the libraries preferablyhave a length between about 10 and 100 base pairs, preferably betweenabout 10 and 75 base pairs, more preferably between about 15 and 50.Probes of longer lengths may also be used.

[0084] Each probe in the library comprises a recognition sequence whichis capable of forming a probe-transcription factor complex with anactivated transcription factor. Due to the high level of DNA bindingspecificity of transcription factors, each transcription factor willtypically bind to a different DNA sequence. In some instances, a relatedfamily of transcription factors may bind to the same DNA sequence.

[0085] By designing the library of probes such that any given probe inthe library includes a DNA recognition sequence which a particularactivated transcription factor (or a related family of activatedtranscription factors) will bind to, and does not also include a DNArecognition sequence which other activated transcription factors willbind to, a given probe may be used to identify a single activatedtranscription factor (or a related family of activated transcriptionfactors).

[0086] It is noted that in certain situations, individual probes maybind to more than one activated transcription factor and may nonethelessbe used in the library. For example, certain probes may bind to arelated family of activated transcription factors. Less than 1:1 bindingspecificity between probes and activated transcription factors can bereadily resolved during analysis of the isolated probes.

[0087] The DNA recognition sequences used in the probes in the librariespreferably have a length between about 10 and 100 base pairs, morepreferably between about 10 and 75 base pairs, more preferably betweenabout 15 and 50 base pairs, more preferably between about 20 and 40 basepairs, and most preferably a length between about 25 and 35 base pairs.

[0088] The optimal length for the recognition sequence and the overallprobe may vary somewhat depending on the particular transcriptionfactor. Hence, one may wish to evaluate the optimal length for therecognition sequence and the probe for a given transcription factorusing a traditional gel shift assay.

[0089] Because the present invention is used to identify knowntranscription factors, it is practical to use longer recognitionsequences in the probes in the library as compared to what would bepractical if a random library were used. For example, at least 1%, 2%,3%, 5%, 10%, 20%, 30%, 50% or more of the probes in the library may haverecognition sequences greater than 35, 40, 45 or more base pairs inlength. By using longer recognition sequences, the probes have greaterbinding specificity and greater binding efficiency to the transcriptionfactors. As a result, the method of the present invention provides ahigh level of sensitivity for isolating probe-transcription factorcomplexes, as described further herein, in combination with a highsignal to noise ratio.

[0090] Selection of which DNA recognition sequences to use in a librarymay be based on the different transcription factors that one wishes todetect in a sample. This, in turn, may depend on the type of organism,cell, or disease state one wishes to identify and/or monitor the geneexpression of. It may also depend on the different functionality thatone wishes to identify or monitor.

[0091] A significant feature of the present invention is the ability todetect multiple different transcription factors at the same time. Thisability arises from the number of different DNA recognition sequencesused in a library, the number of different DNA recognition sequencesrelating directly to the number of different transcription factors thatthe library can be used to detect. A given library of transcriptionfactor probes preferably has at least 2, 3, 5, 10, 20, 50, 100, 250, ormore different DNA recognition sequences. The upper limit on the numberof different DNA recognition sequences that may be incorporated into alibrary is limited only by the number of known DNA recognitionsequences.

[0092] A given library of transcription factor probes may be used todetect gene expression in a single type of cell or organism or may beused to detect gene expression in multiple different types of cells ororganisms. When the library is designed to detect gene expression inmultiple different types of cells or organisms, the library has DNArecognition sequences for multiple different types of cells ororganisms. For example, the library may include DNA recognitionsequences for 2, 3, 4, 5 or more different types of cells or organisms.In one embodiment, the library includes DNA recognition sequences for10, 20, 30, 50, or more different types of cells or organisms.

[0093] If the sample comprises cells that may be from one or moredifferent organisms, the DNA recognition sequences used in the librarymay be for all or some of the different transcription factors expressedby the one or more different organisms. For example, if the library isto be used to classify an unknown type of bacterium, the library mayinclude DNA recognition sequences for multiple different types ofbacteria, thereby allowing the library to be used to classify thebacterium.

[0094] If the sample comprises cells from a particular organism, the DNArecognition sequences used in the library may be for all or some of thedifferent transcription factors expressed by organism. If the library isto be used to classify an unknown type of cells (i.e., determine whethera growth is malignant), the library may include DNA recognitionsequences for multiple different types of cells including the differenttypes of malignant, benign, and normal cell types present in theorganism.

[0095] If the sample comprises cells of a single cell type, the DNArecognition sequences used in the library may be for all or some of thedifferent transcription factors expressed by that cell type. Forexample, if one wishes to monitor the expression of only a particulargroup of genes, such as the genes associated with a particular pathway,the library may include DNA recognition sequences for the transcriptionfactors associated with that group of genes.

[0096] As one can see, a myriad of different libraries of probes can beassembled depending on the nature of the sample and the nature of theanalysis to be performed. It is noted that different libraries may alsobe formed when a particularly large number of transcription factors areto be detected or when different binding conditions are needed fordifferent groups of probes.

[0097] The probes in the library may optionally further comprise adetectable marker which allows the probe to be detected once isolatedfrom the transcription factor probe-transcription factor complex. Sincea wide variety of detection techniques may be used to identify theisolated probes, a similarly wide range of detectable markers may beused in conjunction with those different detection techniques.

[0098] The detectable marker may be any marker which can be used todetermine the presence or absence of the DNA probes. In a preferredembodiment, the detectable marker is biotin and is preferably attachedto one of the 5′ ends of probes. Biotin probes have been found toprovide a desirable high level of sensitivity.

[0099] The detectable marker may also be a dye which can be seen undernatural light or with the assistance of an excitation light source tocause fluorescence. In one embodiment, the detectable marker is afluorescent dye. Examples of fluorescent dyes that may be used include,but are not limited to fluorescein and its derivatives, rhodamine andits derivatives, dansyl, umbeliferone, acridimium, and chemiluminescentmolecules such as luciferin and 2,3-dihydrophthalazinediones. Thefluorescent dye may also be an energy transfer fluorescent dye.

[0100] The detectable marker may also be a molecule which binds to ananalytically detectable counterpart. For example, the detectable markermay be covalently attached to or incorporated into the substrate, forexample, as taught by Ward, European Patent Application No. 63,879. Insuch instances, the substrate is detected by adding the analyticallydetectable counterpart which specifically binds to the substrate,thereby enabling detection of the substrate. Examples of such detectablemarkers and their analytically detectable counterparts include biotinand either fluorescent or chemiluminescent avidin. Antibodies that bindto an analytically detectable antigen may also be used as the detectablemarker. The detectable marker may also be a molecule which, whensubjected to chemical or enzymatic modification, becomes detectable suchas those disclosed in Leary, et al., Proc. Natl. Acad. Sci. (U.S.A.),80:4045-4049 (1983).

[0101] In certain instances, it may be desirable to employ multipledifferent detectable markers. For example, if one wishes to classify anunknown type of cell or organism, the library may include DNA probeswhere different detectable markers are attached to the probes for thedifferent types of cells. Hence, probes for transcription factorsexpressed by malignant cells may include a first fluorescent dye whereasprobes expressed by benign cells may include a second fluorescent dye.In another example, probes for transcription factors expressed by afirst type of lung cancer may include a first fluorescent dye whereasprobes expressed by of a second, different type of lung cancer mayinclude a second fluorescent dye. This allows one to rapidly visuallyidentify the type of cell based on which detectable markers are present.

[0102] When one wishes to compare the gene expression of differentgroups of cells, multiple libraries may be prepared where each librarycontains a different detectable marker. For example, a first librarylabeled with a first detectable marker may be used with a first sampleof cells and a second library labeled with a second detectable markermay be used with a second sample of cells. This way, the isolated probesmay be analyzed together. As described in Section 6A, when an arrayformat is used for detecting the isolated probes, the use of differentdetectable markers is particularly advantageous. For example, as shownin FIG. 3, transcription factor probes isolated from complexes formedfrom a first sample (e.g., a control sample) may have a green dye, andtranscription factor probes isolated from complexes formed from a secondsample (e.g., a test sample) may have a red dye. Regions in the arraywhich are green represent genes which only the cells in the controlsample are expressing, regions in the array which are red representgenes which only cells in the test sample are expressing, and regions inthe array which are both green and red represent genes which cells inboth the control and test samples are expressing.

[0103] 2. Preparation of Sample

[0104] Nuclear extracts can be prepared from a sample of cells using themethod described by Dignam, J. D., Lebovitz, R. M., and Roeder, R. G.(1983). Accurate transcription initiation by RNA polymerase II in asoluble extract from isolated mammalian nuclei. Nucleic Acids Research11:1475-1489. Alternatively, a commercially available kit, such asSigma's Nu-CLEAR Extraction Kit (cat. #N-TRACT) can be used.

[0105] 3. Contacting Cell Sample With Library of Probes

[0106] Once a nuclear extract of a sample of cells is prepared, thenuclear extract is contacted with a library of probes and incubated at15° C. for 30 min.

[0107] 4. Isolation of Probe-Transcription Factor Complexes

[0108] After contacting the sample with the library of transcriptionfactor probes, any probe-activated transcription factor complexes formedare isolated. Any isolation method which can effectively isolate thecomplexes may be used. Isolation is preferably performed by a form ofsize separation, more preferably by electrophoresis, more preferably gelelectrophoresis and most preferably gel electrophoresis using an agarosegel.

[0109] One of the problems overcome by the present invention is theability to isolate complexes of DNA probes bound to transcriptionfactors from other probes in the library. Several methods for performingthis type of isolation were attempted. However, these methods failed toprovide a sufficient yield of probe-transcription factor complexes fromthe sample.

[0110] For example, Applicant attempted to isolate probe-transcriptionfactor complexes by performing an ammonia precipitation. Applicant alsoattempted to isolate probe-transcription factor complexes by passing thesample through a nitrocellulose filter, the filter serving to immobilizeproteins while allowing DNA that is not bound to protein to pass throughthe filter. Unfortunately, neither of these approaches provided asatisfactory yield of complexes for further characterization of theisolated probes.

[0111] Applicant also attempted to isolate probe-transcription factorcomplexes by using acrylamide gel electrophoresis. Unfortunately, thisapproach also did not provide a satisfactory yield of complexes. Withoutbeing bound by theory, it is believed that this method was hampered bythe DNA probes being retained by the acrylamide gel.

[0112] Applicant successfully isolated probe-transcription factorcomplexes from the sample using agarose gel electrophoresis.Interestingly, despite the fact that agarose gel electrophoresis doesnot provide the same quality separation as other forms of gelelectrophoresis (e.g., acrylamide gel electrophoresis), the resolutionprovided using agarose is more than sufficient to effectively separatethe probe-transcription factor complexes. Meanwhile, agarose proved tohave satisfactorily low retention of the DNA probes in the complex,thereby allowing the probes to be further characterized.

[0113] The separated probe-transcription factor complexes were isolatedby removing the band from the gel containing the probe-transcriptionfactor complexes.

[0114] It is noted that simultaneous isolation of multiple differentprobe-transcription factor complexes using a library of DNA probes is asignificant departure from traditional gel-shift assays involving theuse of a single probe to cause the gel shift of a single transcriptionfactor. [Ausebel, F. M. et al eds (1993) Current Protocols in MolecularBiology Vol.2 Greene Publishing Associates, Inc. and John Wiley andSons, Inc., New York]. In such prior gel-shift assays, since only asingle transcription factor was being detected, no efforts were made toisolate the probe used to cause the gel shift. By contrast, in thepresent invention, since multiple probes are used in combination it isnecessary to isolate the complexes in order to isolate and characterizewhich of the plurality of probes in the library formed a complex with atranscription factor and are present in the band.

[0115] It is also noted that simultaneous isolation of multipledifferent probe-transcription factor complexes by isolating andcharacterizing the DNA probes is also a significant departure from U.S.Pat. Nos. 6,066,452 and 5,861,246 and PCT Publication No. WO 00/04196which describe isolation of the nucleic acid binding factors, not theprobes.

[0116] The following is a detailed description of how one may isolateprobe-transcription factor complexes using an agarose gel. It is notedthat other separation and isolation methods may be employed withoutdeparting from the present invention.

[0117] According to one embodiment, a 2% agarose gel in 0.5×TBE isprepared. Preferably, 8 mm-wide combs are used. Each sample is mixedwith 2 μl of a gel loading buffer. Table 1 provides an embodiment of agel loading buffer that may be used. TABLE 1 Gel shift loading buffer:0.25 × TBE buffer: 60% Glycerol: 40% Bromophenol blue: 0.2% (w/v)

[0118] Add 2 ul of gel shift loading buffer and load into a 0.8 cm widthlane of 2% agarose gel in 0.5% TBE. The resulting agarose gel is thenrun in 0.5% TBE at 120V for 16 min. The gel area containing thatcontains the protein/DNA complex, which will be above the blue dye andbelow the gel lane, is excised and transferred to a 1.5 mL tube.

[0119] 5. Separation of Transcription Factor Probes From TranscriptionFactor-Probe Complexes

[0120] Once the transcription factor probe-transcription factorcomplexes are separated from other proteins and DNA in the sample, forexample, as described in Section 4, the transcription factor probes areseparated from the excised portion of the gel.

[0121] The following is a detailed description of how one may isolateprobe-transcription factor complexes from an excised portion of anagarose gel. It is noted that other isolation techniques may be employedwithout departing from the present invention.

[0122] It is noted that the following steps describing the isolation ofthe DNA probes is specifically designed for CLONTECH's NucleoTrap Kit.If another commercially available gel extraction kit is employed, thesteps should be modified in accordance with that manufacturer'sinstructions.

[0123] 1.0 mL of NT1 solution from CLONTECH's NucleoTrap Kit is added tothe excised portion of the gel. The mixture is then incubated at 50° C.until the gel is totally dissolved. The tube is preferably periodicallygently inverted in order to mix the contents until the gel is dissolved.

[0124] 6 μl of beads from a commercially available gel extraction kit isthen preferably added and the resulting mixture is incubated at roomtemperature for 10 min. The tube is preferably periodically gentlyinverted every 2-3 minutes.

[0125] The tube is then microfuged at 10,000 rpm for 30 sec. Theresulting supernatant is carefully removed and the pellet resuspended in150 μl of NT2 solution from CLONTECH's NucleoTrap Kit.

[0126] The pellet is then microfuged at 10,000 rpm for 30 sec. Theresulting supernatant is then carefully removed and the pelletresuspended in 150 μl of NT3 solution from CLONTECH's NucleoTrap Kit.The pellet is then microfuged at 10,000 rpm for 30 sec. The supernatantis again removed and the pellet is allowed to air-dry for 10 min.

[0127] 50 μl of dH₂O is then added to resuspend the pellet. Theresulting mixture is incubated at room temperature for 5 min. Themixture is then gently shaken and incubated for another 5 min.

[0128] The resulting mixture is then microfuged at 10,000 rpm for 1 min.The supernatant is transferred to a fresh 1.5 ml tube. The isolatedsupernatant contains the DNA probes that are bound to proteins. Theisolated supernatant is preferably stored on ice until proceeding to acharacterization of the DNA probes.

[0129] 6. Identifying Isolated Transcription Factor Probes

[0130] A variety of different methods may be used to identify which ofthe transcription factor probes from the library are present in theisolated probe-transcription factor complexes. These methods preferablyalso allow for the amount of transcription factor probes isolated toalso be quantified. By identifying which transcription factor probesform complexes, one is able to determine which transcription factors arepresent in an activated form in the sample, the presence of an activatedtranscription factor evidencing expression of the gene associated withthe transcription factor. By quantifying the amount of eachtranscription factor probe that forms a complex, one is able todetermine the amount of each transcription factor present and hence thelevel of expression of the gene associated with that transcriptionfactor.

[0131] One method that may be used to identify which of thetranscription factor probes from the library are present in the isolatedprobe-transcription factor complexes is mass spectroscopy. According tothis method, the length and composition of each probe can be determined.Therefore, the analyzed results show whether a specific probe isexisting in the complexes and the interactions between transcriptionfactors and binding probes can be determined.

[0132] Another method that may be used to identify which of thetranscription factor probes from the library are present in the isolatedprobe-transcription factor complexes is based on size separation.According to this method, one varies the length of the probes in thetranscription factor probe library so that it is possible to resolve thedifferent sized probes based on a size-based separation. For example,electrophoresis may be performed in order to separate the probes basedon size. Such size-based DNA separations are traditionally done withhigh level specificity for DNA sequencing. By identifying whichtranscription factor probes are present based on the size-basedseparation, one can determine which activated transcription factors arepresent and can also quantify the amount of each activated transcriptionfactor.

[0133] Yet another method for identifying which of the probes from thetranscription factor probe library formed complexes involveshybridization of the transcription factor probes with a hybridizationprobe comprising a complement to the transcription factor probesrecognition sequence. According to this method, detection of aparticular transcription factor probe is accomplished by detecting theformation of a duplex between the transcription factor probe and ahybridization probe comprising a complement to the transcription factorprobe's recognition sequence.

[0134] A wide variety of assays have been developed for performinghybridization assays and detecting the formation of duplexes that may beused in the present invention. For example, hybridization probes with afluorescent dye and a quencher where the fluorescent dye is quenchedwhen the probe is not hybridized to a target and is not quenched whenhybridized to a target oligonucleotide may be used. Suchfluorescer-quencher probes are described in, for example, U.S. Pat. No.6,070,787 and S. Tyagi et al., “Molecular Beacons: Probes that Fluoresceupon Hybridization”, Dept. of Molecular Genetics, Public Health ResearchInstitute, New York, N.Y., Aug. 25, 1995, each of which are incorporatedherein by reference. By attaching different fluorescent dyes todifferent hybridization probes, it is possible to determine whichtranscription factor probes from the library formed complexes based onwhich fluorescent dyes are present (e.g., fluorescent dye and quencheron hybridization probe or fluorescent dye on hybridization probe andquencher on transcription factor probe). Applicant notes that one mayalso attach different fluorescent dyes to different transcription factorprobes and use a change in fluorescence due to hybridization to ahybridization probe to determine which transcription factor probes arepresent (e.g., fluorescent dye and quencher on transcription factorprobe or fluorescent dye on transcription factor probe and quencher onhybridization probe).

[0135] A difficulty, however, arises when using multiple differentfluorescer to detect multiple different transcription factor probes.Namely, there is a limited number of different fluorescers that may bespectrally resolved. As a result, a limited number of differenttranscription factors can be detected at the same time, for example onlyas many as five to ten.

A. Hybridization Arrays For Detecting Isolated Transcription FactorProbes

[0136] A preferred assay for detecting the formation of duplexes betweentranscription factor probes and hybridization probes comprising theircomplements involves the use of an array of hybridization probesimmobilized on a solid support. The hybridization probes comprisesequences that are complementary to at least a portion of therecognition sequences of the transcription factor probes and thus areable to hybridize to the different probes in a transcription factorprobe library.

[0137] In order to improve enhance the sensitivity of the hybridizationarray, the immobilized probes preferably provide at least 2, 3, 4 ormore copies of at least a portion of the recognition sequenceincorporated into the transcription factor probes.

[0138] According to the present invention, the hybridization probesimmobilized on the array preferably are at least 25 nucleotides inlength, more preferably at least 30, 40 or 50 nucleotides in length. Theimmobilized hybridization probes may be 50, 75, 100 nucleotides orlonger in length. In one preferred embodiment the immobilized probes arebetween 50 and 100 nucleotides in length.

[0139] By immobilizing hybridization probes on a solid support whichcomprise one or more copies of a complement to at least a portion of therecognition sequences of the transcription factor probes, thehybridization probes serve as immobilizing agents for the transcriptionfactor probes, each different hybridization probe being designed toselectively immobilize a different transcription factor probe.

[0140]FIG. 2 illustrates an array of hybridization probes attached to asolid support where different hybridization probes are attached todiscrete, different regions of the array. Each different region of thearray comprises one or more copies of a same hybridization probe whichincorporates a sequence that is complementary to a recognition sequenceof a transcription factor probe. As a result, the hybridization probesin a given region of the array can selectively hybridize to andimmobilize a different transcription factor probe based on thetranscription factor probe's recognition sequence.

[0141] By detecting which regions the isolated transcription factorprobes hybridize to on the array, one can determine which activatedtranscription factors are present in the sample and can also quantifythe amount of each activated transcription factor.

[0142] These arrays can be designed and used to study transcriptionfactor activation in a variety of biological processes, including cellproliferation, differentiation, transformation, apoptosis, drugtreatment, and others described herein.

[0143] Numerous methods have been developed for attaching hybridizationprobes to solid supports in order to perform immobilized hybridizationassays and detect target oligonucleotides in a sample. Numerous methodsand devices are also known in the art for detecting the hybridization ofa target oligonucleotide to a hybridization probe immobilized in aregion of the array. Examples of such methods and device for formingarrays and detecting hybridization include, but are not limited to thosedescribed in U.S. Pat. Nos. 6,197,506, 6,045,996, 6,040,138, 5,424,186,5,384,261, each of which are incorporated herein by reference.

[0144] Several modifications may be made to the hybridization arraysknown in the art in order to customize the hybridization arrays for usein detecting activated transcription factors through thecharacterization of isolated transcription factor probes which form acomplex with the activated transcription factors.

[0145] Since the hybridization probe arrays of the present invention aredesigned to hybridize to the probes in the transcription factor probelibrary by comprising a sequence that is complementary to thetranscription factor recognition sequence, the composition of thehybridization probes in the array should complement the recognitionsequences of the probes in the transcription factor probe library. Asdiscussed in Section 1, a variety of different libraries oftranscription factor probes are provided that may be used to detectactivated transcription factors according to the present invention.

[0146] Selection of the sequences used in the hybridization probes maybe based on the different transcription factors that one wishes todetect in a sample. This, in turn, may depend on the type of organism,cell, or disease state one wishes to identify and/or monitor the geneexpression of.

[0147] A significant feature of the present invention is the ability todetect multiple different transcription factors at the same time. Thisability arises from the number of different DNA recognition sequencesused in a library, the number of different DNA recognition sequencesrelating directly to the number of different transcription factors thatthe library can be used to detect. A given array of hybridization probespreferably has complements for at least 2, 3, 5, 10, 20, 30, 50, 100,250 or more different DNA recognition sequences. The upper limit on thenumber of different DNA recognition sequences that the array ofhybridization probes may detect is limited only by the number of knownDNA recognition sequences and hence the number of known complements tothe DNA recognition sequences.

[0148] A given array of hybridization probes may be used to detect geneexpression in a single type of cell or organism or may be used to detectgene expression in multiple different types of cells or organisms. Whenthe array is designed for use with a library designed to detect geneexpression in multiple different types of cells or organisms, the arraymay include complements to DNA recognition sequences for multipledifferent types of cells or organisms. For example, the array mayinclude complements to DNA recognition sequences for 2, 3, 4, 5 or moredifferent types of cells or organisms. In one embodiment, the array mayinclude complements to DNA recognition sequences for 10, 20, 30, 50, ormore different types of cells or organisms.

[0149] If the sample to be analyzed comprises cells that may be from oneor more different organisms, the DNA recognition sequences used in thelibrary may be for all or some of the different transcription factorsexpressed by the one or more different organisms. For example, iflibrary is to be used to classify an unknown type of bacterium, thelibrary may include DNA recognition sequences for multiple differenttypes of bacteria, thereby allowing the library to be used to classifythe bacterium. Accordingly, the array used in combination with thelibrary would include complements to DNA recognition sequences formultiple different types of bacteria.

[0150] If the sample comprises cells from a particular organism, the DNArecognition sequences used in the library may be for all or some of thedifferent transcription factors expressed by organism. Accordingly, thearray used in combination with the library would include complements toDNA recognition sequences for the different transcription factorsexpressed by organism.

[0151] If the library is to be used to classify an unknown type of cells(i.e., determine whether a growth is malignant), the library may includeDNA recognition sequences for multiple different types of cellsincluding the different types of malignant, benign, and normal celltypes present in the organism. Accordingly, the array used incombination with the library would include complements to DNArecognition sequences for the multiple different types of cells.

[0152] If the sample comprises cells of a single cell type, the DNArecognition sequences used in the library may be for all or some of thedifferent transcription factors expressed by that cell type.Accordingly, the array used in combination with the library wouldinclude complements to the recognition sequences for all or some of thedifferent transcription factors expressed by that cell type.

[0153] i. Procedure for Performing Hybridization Using Array

[0154] Provided below is a description of a procedure that may be usedto hybridize isolated transcription factor probes to a hybridizationarray. It is noted that the below procedure may be varied and modifiedwithout departing from other aspects of the invention.

[0155] An array membrane having hybridization probes attached for thetranscription factor probes is first placed into a hybridization bottle.The membrane is then wet by filling the bottle with deionized H₂O. Afterwetting the membrane, the water is decanted. Membranes that may be usedas array membranes include any membrane to which a hybridization probemay be attached. Specific examples of membranes that may be used asarray membranes include, but are not limited to NYTRAN membrane(Schleicher & Schuell), BIODYNE membrane (Pall), and NYLON membrane(Roche Molecular Biochemicals).

[0156] 5 ml of prewarmed hybridization buffer is then added to eachhybridization bottle containing an array membrane. The bottle is thenplaced in a hybridization oven at 42° C. for 2 hr. An example of ahybridization buffer that may be used is EXPHYP by Clonetech.

[0157] After incubating the hybridization bottle, a thermal cycler maybe used to denature the hybridization probes by heating the probes at90° C. for 3 min, followed by immediately chilling the hybridizationprobes on ice.

[0158] The isolated probe-transcription factors complexes are then addedto the hybridization bottle. Hybridization is preferably performed at42° C. overnight.

[0159] After hybridization, the hybridization mixture is decanted fromthe hybridization bottle. The membrane is then washed repeatedly.

[0160] In one embodiment, washing includes using 60 ml of a prewarmedfirst hybridization wash which preferably comprises 2×SSC/0.5% SDS. Themembrane is incubated in the presence of the first hybridization wash at42° C. for 20 min with shaking. The first hybridization wash solution isthen decanted and the membrane washed a second time. A secondhybridization wash, preferably comprising 0.1×SSC/0.5% SDS is then usedto wash the membrane further. The membrane is incubated in the presenceof the second hybridization wash at 42° C. for 20 min with shaking. Thesecond hybridization wash solution is then decanted and the membranewashed a second time.

[0161] ii. Procedure for Detecting Array Hybridization

[0162] The following describes a procedure that may be used to detectisolated transcription factor probes isolated on the hybridizationarray. It is noted that each membrane should be separately hybridized,washed and detected in separate containers in order to prevent crosscontamination between samples. It is also noted that it is preferredthat the membrane is not allowed to dry during detection.

[0163] According to the procedure, the membrane is carefully removedfrom the hybridization bottle and transferred to a new containercontaining 30 ml of 1× blocking buffer. The dimensions of each containeris preferably about 4.5″×3.5″, equivalent in size to a 200 μLpipette-tip container. Table 2 provides an embodiment of a blockingbuffer that may be used. TABLE 2 1 × Blocking Buffer: Blocking reagent:1%  0.1M Maleic acid 0.15M NaCl Adjusted with NaOH to pH 7.5

[0164] It is noted that the array membrane may tend to curl adjacent itsedges. It is desirable to keep the array membrane flush with the bottomof the container.

[0165] The array membrane is incubated at room temperature for 30 minwith gentle shaking. 1 ml of blocking buffer is then transferred fromeach membrane container to a fresh 1.5 ml tube. 3 μl of Streptavidin-APconjugate is then added to the 1.5 ml tube and is mixed well. Thecontents of the 1.5 ml tube is then returned to the container and thecontainer is incubated at room temperature for 30 min.

[0166] The membrane is then washed three times at room temperature with40 ml of 1× detection wash buffer, each 10 min. Table 3 provides anembodiment of a 1× detection wash buffer that may be used. TABLE 3 1 ×Detection wash buffer:  10 mM Tris-HCl, pH 8.0 150 mM NaCl 0.05%Tween-20

[0167] 30 ml of 1× detection equilibrate buffer is then added to eachmembrane and the combination is incubated at room temperature for 5 min.Table 4 provides an embodiment of a 1× detection equilibrate buffer thatmay be used. TABLE 4 1 × Detection equilibrate buffer: 0.1 M Tris-HCl pH9.5 0.1 M NaCl

[0168] The resulting membrane is then transferred onto a transparencyfilm. 3 ml of CPD-Star substrate, produced by Applera, AppliedBiosystems Division, is then pipetted onto the membrane.

[0169] A second transparency film is then placed over the firsttransparency. It is important to ensure that substrate is evenlydistributed over the membrane with no air bubbles. The sandwich oftransparency films are then incubated at room temperature for 5 min.

[0170] The CPD-Star substrate is then shaken off and the films arewiped. The membrane is then exposed to Hyperfilm ECL, available fromAmersham-Pharmercia. Alternatively, a chemiluminescence imaging systemmay be used such as the ones produced by ALPHA INNOTECH. It may bedesirable to try different exposures of varying lengths of time (e.g.,2-10 min).

[0171] The hybridization array may be used to obtain a quantitativeanalysis of the amount of transcription factor probe present. Forexample, if a chemiluminescence imaging system is being used, theinstructions that come with that system's software should be followed.If Hyperfilm ECL is used, it may be necessary to scan the film to obtainnumerical data for comparison.

[0172] iii. Normalization of Data From Array Hybridization

[0173] One of the advantages provided by array hybridization fordetecting isolated transcription factor probes is the ability tosimultaneously analyze whether multiple different activatedtranscription factors are present.

[0174] A further advantage provided is that the system allows one tocompare a quantification of multiple different activated transcriptionfactors between two or more samples. When two or more arrays frommultiple samples are compared, it is desirable to normalize them.

[0175] In order to facilitate normalization of the arrays, an internalstandard may be used so that the intensity of detectable marker signalsbetween arrays can be normalized. In certain instances, the internalstandard may also be used to control the time used to develop thedetectable marker.

[0176] In one embodiment, the internal standard for normalization isbiotinylated DNA which is spotted on a portion of the array, preferablyadjacent one or more sides of the array. For example, biotin-labeledubiquitin DNA may be positioned on the bottom line and last column ofthe array. In order to normalize two or more arrays for comparison ofresults, the exposure time for each array should be adjusted so that thesignal intensity in the region of the biotinylated DNA is approximatelyequivalent on both arrays.

[0177] 7. Use of Multiple Libraries In Combination To Compare GeneExpression Between Different Samples

[0178] When an array format is used for detecting the isolated probes,it may be desirable to use multiple libraries labeled with differentdetectable markers in order to facilitate comparison between samples.For example, as shown in FIG. 3, probes from a first sample (e.g., acontrol sample) may have a green dye, and probes from a second sample(e.g., a test sample) may have a red dye. Both probes are separated fromtheir bound complexes, mixed, and hybridized to a single array. Greenspots in the array represent genes which only the cells in the controlsample are expressing, and red spots in the array represent genes whichonly cells in the test sample are expressing. When both dyes hybridizeto the same spot in an equal amount, the balanced mixture of green andred appears as yellow in the array, representing genes which cells inboth the control and test samples are expressing.

[0179] One embodiment of this application of the present invention thusrelates to a method for comparing gene expression between a test sampleand a control sample, the method comprising forming transcription factorprobe-activated transcription factors complexes using the test sampleand a first library of transcription factor probes having a firstdetectable marker; forming transcription factor probe-activatedtranscription factors complexes using the control sample and a secondlibrary of transcription factor probes having the same nucleic acidsequences as the first library but having a second, different detectablemarker; isolating the transcription factor probes from the first librarywhich formed complexes involving transcription factors from the testsample; isolating the transcription factor probes from the secondlibrary which formed complexes involving transcription factors from thecontrol sample; and detecting the isolated transcription factor probesfrom the first and second libraries using a same hybridization array.

[0180] Another embodiment of this application of the present inventionrelates to a kit comprises the first and second library of transcriptionfactor probes. The kit may optionally further include a hybridizationarray comprising complements to the transcription factor probes. The kitmay also include instructions for isolating the transcription factorprobe-activated transcription factor complexes using agarose gel.

[0181] 8. Applications For Monitoring Gene Expression Via Detection OfActivated Transcription Factors

[0182] By better understanding which cells express which genes and howdifferent conditions influence gene expression, fundamental questions ofbiology can be answered. Thus, by being able to rapidly and efficientlydetect multiple activated transcription factors at the same time, thepresent invention avails itself to numerous valuable applicationsrelating to the monitoring of gene expression. Some of theseapplications are described herein. Other applications will be apparentto those of ordinary skill.

[0183] A. Characterization of Cell Type

[0184] By detecting and optionally quantifying which activatedtranscription factors are present in a cell sample, the methods of thepresent invention allow one to identify which genes are being expressedand to what extent each gene is being expressed. Different types ofcells for a particular organism will express different genes. As aresult, the present invention allows one to rapidly characterize a celltype based on which activated transcription factors are present and atwhat levels.

[0185] One embodiment of this application of the present invention thusrelates to a method for characterizing a cell type, the methodcomprising forming transcription factor probe-activated transcriptionfactors complexes using a test sample and a library of transcriptionfactor probes comprising recognition sequences characteristic ofdifferent types of cells; isolating the transcription factor probes fromthe library which formed complexes involving transcription factors fromthe test sample; and detecting the isolated transcription factor probesusing a hybridization array comprising sequences complementary to thetranscription factor probes in the library.

[0186] A further embodiment of this application of the present inventionrelates to a library of transcription factor probes and hybridizationarray comprising complements to the library of transcription factorprobes are provided where the transcription factor probes compriserecognition sequences from multiple different cell types. A kit is alsoprovided that comprises both the library of probes and the hybridizationarray. The kit may also include instructions for isolating thetranscription factor probe-activated transcription factor complexesusing an agarose gel, either in combination with the library, thehybridization array, or both.

[0187] It is noted that different organisms will also express differentactivated transcription factors. Characterizing the mixture of differentactivated transcription factors expressed by a particular organism(e.g., a culture of bacteria) can be used to identify the particularorganism. This application of the method of the present invention may beparticularly useful for rapidly characterizing microbes such as bacteriaand diseased tissue.

[0188] One embodiment of this application of the present invention thusrelates to a method for characterizing an organism, the methodcomprising forming transcription factor probe-activated transcriptionfactors complexes using a test sample and a library of transcriptionfactor probes comprising recognition sequences characteristic ofdifferent organisms; isolating the transcription factor probes from thelibrary which formed complexes involving transcription factors from thetest sample; and detecting the isolated transcription factor probesusing a hybridization array comprising sequences complementary to thetranscription factor probes in the library.

[0189] A further embodiment of this application of the present inventionrelates to a library of transcription factor probes and hybridizationarray comprising complements to the library of transcription factorprobes are provided where the transcription factor probes compriserecognition sequences from multiple different organisms. A kit is alsoprovided that comprises both the library of probes and the hybridizationarray. The kit may also include instructions for isolating thetranscription factor probe-activated transcription factor complexesusing an agarose gel, either in combination with the library, thehybridization array, or both.

[0190] It is noted that the mixture of different activated transcriptionfactors expressed by different cell types or organisms may be usedaccording to the present invention as a form of an expression signaturefor that cell type. FIG. 2 illustrates an array detection format. Thepattern formed by the detectable markers [cubes] in FIG. 2 can be usedas a visual fingerprint of the expression signature and can be used toidentify a particular cell type or organism based on that visualfingerprint. In this regard, it is envisioned that an array may bedeveloped with a great multiplicity of immobilizing agents for differenttranscription factor probes. It is noted that the number of cubes shownin the figure is employed to reflect signal intensity.

[0191] The array may include immobilizing agents for transcriptionfactor probes for different cell types and/or for different organisms.By comparing the array pattern to a standard for a particular cell typeor organism, the cell type or organism can be rapidly determined.

[0192] B. Determining the Functions of Different Genes

[0193] Despite the fact that each cell in the human body contains thesame set of genes, the human body is comprised of a wide diversity ofdifferent cell types that work in concert to form the human body. Thewide diversity of cell types present in the human body and othermulticellular organisms is due to variations between cells regardingwhich genes are expressed, the level at which the genes are expressed,and the conditions under which the genes are expressed. The presentinvention provides the unique ability of rapidly determining which of agreat number of genes are expressed by numerous different cell types. Bybeing able to determine which genes are expressed by which cell types,the functions of different genes can be deduced.

[0194] C. Diagnosis of Disease States

[0195] Certain disease states may be caused and/or characterizable bycertain genes being expressed or not expressed as compared to normalcells. Other disease states may result from and/or be characterizable bycertain genes being transcribed at different levels as compared tonormal cells.

[0196] By being able to rapidly monitor the expression levels ofmultiple different genes, the present invention provides an accuratemethod for diagnosing certain disease states known to be associated withthe expression non-expression, reduced expression, and/or elevatedexpression of one or more genes. Conversely, by comparing the expressionnon-expression, reduced expression, and/or elevated expression of one ormore genes in normal and abnormal cells, present invention facilitatesthe association of one or more genes with certain disease states. Byunderstanding that a particular disease state is caused by a differentexpression (higher or lower) of one or more proteins, it should bepossible to remedy the disease state by increasing or decreasing theexpression of the one or more proteins, by administering the one or moreproteins or, if particular proteins are overexpressed, by inhibiting theone or more proteins.

[0197] One embodiment of this application of the present invention thusrelates to a method for diagnosing a disease state of a sample of cells,the method comprising forming transcription factor probe-activatedtranscription factors complexes using the sample of cells and a libraryof transcription factor probes comprising different transcription factorrecognition sequences; isolating the transcription factor probes fromthe library which form complexes involving transcription factors fromthe sample; detecting the isolated transcription factor probes using ahybridization array comprising sequences complementary to thetranscription factor probes in the library; and diagnosing a presence ofa disease state based on which transcription factors are activated inthe cell sample as identified by which transcription factor probes areisolated.

[0198] D. Compound Screening For Drug Candidates

[0199] Being able to monitor transcription factor activity for multipledifferent transcription factors at the same time is of great importanceto developing a better understanding of different roles that varioustranscription factors play. In addition, monitoring multiple differenttranscription factors at the same time allows one to rapidly screen forcompounds that influence transcription factor activity, referred toherein as a “transcription factor modulator.”

[0200] The present invention may thus be used as a high throughputscreening assay for transcription factor modulators that either up- ordown-regulate genes by influencing the synthesis and activation oftranscription factors for those genes.

[0201] By having a further understanding of what compounds modulatetranscription factor activity, such compounds may be more effectivelyused for in vitro modification of signal transduction, transcription,splicing, and the like, e.g., as tools for recombinant methods, cellculture modulators, etc. More importantly, such compounds can be used aslead compounds for drug development for a variety of conditions,including as antibacterial, antifungal, antiviral, antineoplastic,inflammation modulatory, or immune system modulatory agents.Accordingly, being able to monitor transcription factor activity formultiple different factors has great use for screening compounds toidentify lead compounds for pharmaceutical or other applications.

[0202] Indeed, because gene expression is fundamental in all biologicalprocesses, including cell division, growth, replication,differentiation, repair, infection of cells, etc., the ability tomonitor transcription factor activity and identify compounds whichmodulator their activity can be used to identify drug leads for avariety of conditions, including neoplasia, inflammation, allergichypersensitivity, metabolic disease, genetic disease, viral infection,bacterial infection, fungal infection, or the like. In addition,compounds which specifically target transcription factors in undesiredorganisms such as viruses, fungi, agricultural pests, or the like, canserve as fungicides, bactericides, herbicides, insecticides, and thelike. Thus, the range of conditions that are related to transcriptionfactor activity includes conditions in humans and other animals, and inplants, e.g., for agricultural applications.

[0203] As used herein, the term “transcription factor modulator” refersto any molecule or complex of more than one molecule that affects theregulatory region. The present invention contemplates screens forsynthetic small molecule agents, chemical compounds, chemical complexes,and salts thereof as well as screens for natural products, such as plantextracts or materials obtained from fermentation broths. Other moleculesthat can be identified using, the screens of the invention includeproteins and peptide fragments, peptides, nucleic acids andoligonucleotides (particularly triple-helix-forming oligonucleotides),carbohydrates, phospholipids and other lipid derivatives, steroids andsteroid derivatives, prostaglandins and related arachadonic acidderivatives, etc.

[0204] Existing methods for monitoring gene expression typically monitordown-stream expression processes by measuring mRNA or the resulting geneproduct. However, why a particular mRNA or protein is expressed athigher or lower levels is not revealed by these methods. This is becausea given compound can influence the formation of a transcription factor,influence the activation of the transcription factor, interact with theactivated transcription factor, interact with the regulatory element towhich the transcription factor binds, or interact with the mRNA that isproduced.

[0205] By contrast, because the present invention is specific todetecting activated transcription factors, the present invention can beeffectively used to screen for drugs that have a mechanism of actiondirectly related to the expression and/or activation of transcriptionfactors.

[0206] It should be noted that methods exist for measuring atranscription factor in a sample. However, because such methods detectthe protein itself, they are unable to determine whether thetranscription factor is activated, i.e., it is capable of binding to aregulatory element. By being able to detect whether multiple differenttranscription factors are activated, the present invention, when used incombination with an assay for detecting the amount of activated andunactivated transcription factor, allows one to evaluate specificallyhow a given compound influences the activation of differenttranscription factors.

[0207] The present invention may be used to screen large chemicallibraries for modulator activity for multiple different transcriptionfactors. For example, by exposing cells to different members of thechemical libraries, and performing the methods of the present invention,one is able to screen the different members of the library relative tomultiple different transcription factors at the same time.

[0208] It will be appreciated that there are many suppliers of chemicalcompounds, including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs Switzerland) and the like.

[0209] In one preferred embodiment, high throughput screening involvestesting a combinatorial library containing a large number of potentialmodulator compounds. A combinatorial chemical library may be acollection of diverse chemical compounds generated by either chemicalsynthesis or biological synthesis, by combining a number of chemical“building blocks” such as reagents. For example, a linear combinatorialchemical library such as a polypeptide library is formed by combining aset of chemical building blocks (amino acids) in every possible way fora given compound length (i.e., the number of amino acids in apolypeptide compound). Millions of chemical compounds can be synthesizedthrough such combinatorial mixing of chemical building blocks.

[0210] Such combinatorial libraries are then screened to identify thoselibrary members (particular chemical species or subclasses) thatmodulate one or more transcription factors. The compounds thusidentified can serve as conventional “lead compounds” or can themselvesbe used as potential or actual therapeutics for the one or moretranscription factors whose activities the compounds modulate.

[0211] Preparation and screening of combinatorial libraries is wellknown to those of skill in the art. Such combinatorial librariesinclude, but are not limited to, peptide libraries (e.g., U.S. Pat. No.5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493 (1991) andHoughton et al., Nature 354:84-88 (1991)). Other chemistries forgenerating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (PCT PublicationNo. WO 91/19735), encoded peptides (PCT Publication WO 93/20242), randombio-oligomers (PCT Publication No. WO 92/00091), benzodiazepines (U.S.Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines anddipeptides (Hobbs et al., Proc. Nat. Acad. Sci. USA 90:6909-6913(1993)), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc.114:6568 (1992)), nonpeptidal peptidomimetics with .beta.-D-glucosescaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218(1992)), analogous organic syntheses of small compound libraries (Chenet al., J. Amer. Chem. Soc. 116:2661 (1994)), oligocarbamates (Cho etal., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell etal., J. Org. Chem. 59:658 (1994)), nucleic acid libraries (see, Ausubel,Berger and Sambrook, all supra), peptide nucleic acid libraries (see,e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn etal., Nature Biotechnology, 14(3):309-314 (1996) and PCT/US96/10287),carbohydrate libraries (see, e.g., Liang et al., Science, 274:1520-1522(1996) and U.S. Pat. No. 5,593,853), small organic molecule libraries(see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993);isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones andmetathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos.5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337;benzodiazepines, 5,288,514, and the like).

[0212] Devices for the preparation of combinatorial libraries are alsocommercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech,Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A AppliedBiosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, numerous combinatorial libraries are themselvescommercially available (see, e.g., ComGenex, Princeton, N.J., Asinex,Moscow, Ru, Tripos, Inc., St. Louis, Mo., ChemStar, Ltd, Moscow, RU, 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

[0213] Control reactions may be performed in combination with thelibraries. Such optional control reactions are appropriate and increasethe reliability of the screening. Accordingly, in a preferredembodiment, the methods of the invention include such a controlreaction. The control reaction may be a negative control reaction thatmeasures the transcription factor activity independent of atranscription modulator. The control reaction may also be a positivecontrol reaction that measures transcription factor activity in view ofa known transcription modulator.

[0214] By being able to screen multiple different transcription factorsat the same time, not only is it possible to screen a large number ofpotential transcription modulators per day, it is also possible toscreen any potential transcription modulator relative to a large numberof different transcription factors. The ability to screen multipledifferent transcription factors at the same time thus greatly enhancesthe high throughput capabilities of this screening assay.

[0215] E. Evaluation of Drug Efficacy

[0216] Given that certain disease states may be caused by an unusuallevel of transcription of one or more genes, drugs may be designed toeither stimulate or inhibit transcription in order make gene expressionof diseased cells approach the gene expression of normal cells. A rapidand effective method for monitoring gene expression is thus highlyadvantageous for evaluating the effectiveness of a drug's ability toalter the transcription of one or more genes. The effectiveness of adrug being delivered to a site of action as well as the drug's efficacyin vivo can thus be evaluated with the assistance of the methods of thepresent invention.

[0217] Also of great concern when developing new drugs is the sideeffects which the drugs might have. One approach for screening drugcandidates for undesirable side effects would be to employ the presentinvention to monitor how gene expression is altered in response to theadministration of a drug candidate. By understanding how a candidateaffects gene expression, candidates likely to have undesirable sideaffects can be rapidly identified.

[0218] Because the biological importance of transcription factors, theyare ideal drug targets. Traditional transcription factor screeningassays only detect one transcription factor at a time. As a result,existing assays are tremendously in efficient for detecting how a drugeffects different gene expression. However, with the assistance of thepresent invention, it is now possible to screen hundreds and eventhousands of transcription factors in a short amount of time in order tomonitor how a given drug affects the expression of wide range of genes.The present invention will thus dramatically facilitate the screeningprocess of identifying new drugs, characterizing their mechanism of theaction, and screening for adverse side effects based on the drug'simpact on expression.

[0219] 9. Determining Sequence Binding Requirements For TranscriptionFactors

[0220] In general, a further application of the present invention is therapid and efficient determination of the DNA sequence bindingrequirements for a given transcription factor. By being able toefficiently isolate DNA probes from multiple DNA probe-transcriptionfactor complexes, the present invention makes it feasible to identifywhich DNA sequences bind to transcription factors, quantify the amountof each DNA sequence isolated, and use that information to determinedifferent DNA sequence binding requirements for a given transcriptionfactor in a high throughput manner.

[0221] As a result of the completion of the human genome project, manynew proteins will be discovered. Based on their primary structure,transcription factors for these proteins can be identified. However, theidentification of DNA sequences to which these transcription factorswill bind is a more difficult problem which the present invention helpsto address.

[0222] When determining the DNA sequences to which transcription factorsbind, there are several different questions that need to be answered.One question relates to the identification of an optimal bindingsequence for the transcription factor. Another question relates to theidentification of the minimal sequence required for binding. Yet anotherquestion which is related to the prior question, relates to theidentification of a consensus sequence which is the minimum sequencerequired for binding. The present invention can be used to facilitatethe answering of each of these questions.

[0223] The present invention enables one to rapidly determine a set ofDNA sequences to which an activated transcription factor will bind and aset of DNA sequences to which an activated transcription factor will notbind. The amount isolated of each member in the set of DNA sequences towhich an activated transcription factor binds may also be quantified.

[0224] Using this information, the minimum sequence required for thetranscription factor to bind may be determined. Varying degrees ofconsensus sequences among the DNA sequences to which an activatedtranscription factor binds can also be determined. Meanwhile, byquantifying the amount of isolated DNA, how different base substitutionsaffect transcription factor binding can also be evaluated. This enablesone to determine the sequences to which a given transcription factorbinds most tightly.

[0225]FIG. 4 illustrates a process whereby the minimum DNA sequencebinding requirements for a given transcription factor can be rapidlydetermined. As illustrated, a sample containing an activatedtranscription factor is contacted with a library of probes. The probesin the library comprise a DNA sequence and a detectable marker.

[0226] Since, in this embodiment, the library of probes are used todetermine the minimum DNA sequence binding requirements, the DNAsequences used in the library may be variations on a sequence which thetranscription factor is known to bind to. Alternatively, the sequencesused in the library may selected without knowledge of the bindingspecificity of the activated transcription factor.

[0227] If the DNA probes used to perform this method are known, a simplehybridization array having complements to the DNA probes in the librarymay be employed, as described above. However, if a random library of DNAprobes is employed, any isolated DNA probes can be characterized byexisting position-fixed DNA array technology.

[0228] As a result of contacting the sample with the library of probes,complexes are formed between the activated transcription factor presentin the sample and DNA probes in the library which have sequences thatsatisfy the sequence specificity of the DNA-binding domain of theactivated transcription factor. The resulting probe-transcription factorcomplexes are preferably isolated by purification from agarose gel asdescribed previously. After isolating the DNA probe-activatedtranscription factor complexes, those probes from the library which bindto transcription factors in the sample may be further isolated andcharacterized as discussed previously.

[0229] Since only those probes from the library which form a complexwith an activated transcription factor will be isolated, identificationof which probes are isolated serves to identify the range of sequencesto which the activated transcription factor is capable of binding.

[0230] By constructing a consensus sequence based on the isolatedprobes, one is able to more precisely define the minimum bindingrequirements for the transcription factor. Furthermore, once a consensussequence and a series of binding sequences are known, this informationcan be used to locate the occurrence of those sequences in 5′untranslation regions within a genome. This will allow researchers toidentify which proteins may be regulated by the transcription factor.Genomes of different organisms may also be researched to identifyproteins that may be functionally related.

[0231] Depending on the level of diversity of the library used, afurther round of screening may be used to map the binding requirementsof the transcription factor in greater detail. For example, when one ormore binding sequences are identified, further experimentation may alsobe conducted to identify more binding sequences. This may involvecreating a library of random mutations based on one or more DNAsequences shown to bind to the transcription factor in the prior screen.Binding of the mutants may be performed in order to identify othermutants to which the transcription factor binds. Multiple cycles ofgenerating and screening mutant libraries may be conducted as isnecessary and desirable.

[0232]FIG. 5 illustrates a variation of the method described in regardto FIG. 4 where an optimal sequence for binding is identified. Asillustrated in FIG. 5, the isolated DNA probes are quantified as well asidentified. By monitoring how changes in the sequence affect the amountof each probe isolated, one is able to see how the different sequencescompete for binding to the transcription factor. As a result, an optimaltranscription factor binding sequence can be identified.

[0233] Depending on the level of diversity of the library used toperform the first screen, when one or more binding sequences areidentified, further experimentation may also be conducted to identify abetter binding sequence. This may involve creating a library of randommutations based on one or more DNA sequences shown to bind to thetranscription factor in the prior screen. Quantification of the bindingof the mutants may be performed in order to identify a stronger bindingmutant. Multiple cycles of generating and screening mutant libraries maybe conducted as is necessary and desirable.

[0234] 10. Determining Transcription Factor Expression and Activation

[0235] Prior to being activated, a transcription factor must beexpressed. However, not all expressed transcription factors areactivated. By determining whether multiple different transcriptionfactors are being activated according to the present invention, incombination with determining whether the different transcription factorsare being expressed, the present invention provides a rapid andefficient method for monitoring how different transcription factorexpression and transcription factor activation change.

[0236] One application of this method relates to the diagnosis ofdisease states. By being able to track both changes in transcriptionfactor expression and activation, more precise diagnosis of the cause ofgenetically related diseases may be discovered.

[0237] A further application of this method relates to the evaluation ofhow different agents or conditions affect both transcription factorexpression and activation. For example, different agents can be screenedfor their ability to inhibit and/or activate the expression of certainproteins or impact different disease states. By knowing how the agentaffects both transcription factor expression and activation, one is ableto identify the mode of action of the agent. By being able to screenmultiple transcription factors at the same time, one is further able toscreen those agents for otherwise unforeseen adverse affects on thegenetic level.

[0238] 11. Kits For Use In The Present Invention

[0239] A wide variety of kits may be designed for use with the presentinvention. Various examples of these kits have already been describedand additional kits are further described herein.

[0240] In one particular embodiment, a kit is provided which includes alibrary of transcription factor probes comprising recognition sequencesfor transcription factors. The kit further includes an array ofhybridization probes where each probe comprises a sequence that iscomplementary to at least a portion of the recognition sequences on theprobes in the library. In a preferred embodiment, the probes in thearray of hybridization probes comprise 2, 3, 4, or more copies of asequence that is complementary to at least a portion of the recognitionsequences on the probes in the library.

[0241] In another particular embodiment, a kit is provided whichincludes a first library of transcription factor probes comprisingrecognition sequences for transcription factors, each probe in the firstlibrary further comprising a first detectable marker. The kit furtherincludes a second library of transcription factor probes comprising thesame recognition sequences for transcription factors as the firstlibrary, each probe in the second library further comprising a seconddetectable marker that is different than the first detectable marker.

[0242] In another particular embodiment, a kit is provided whichincludes a library of transcription factor probes comprising recognitionsequences for transcription factors in combination with instructions forusing agarose gel to isolate transcription factor probe-transcriptionfactor complexes.

[0243] In yet another embodiment, a kit is provided which includes anarray of hybridization probes where each probe comprises a sequence thatis complementary to at least a portion of recognition sequences oftranscription factors. The kit further includes instructions for usingagarose gel to isolate transcription factor probe-transcription factorcomplexes.

[0244] In yet another embodiment, a kit is provided which includes ahybridization array according to the present invention, hybridizationbuffer, detection wash buffer, and detection equilibrate buffer.

[0245] With regard to any of the kit embodiments, it is noted that thelibraries of transcription factor probes and hybridization arrays may bevaried as has been described herein.

[0246] 12. Array Detection of Different Activated Transcription Factors

[0247] This section describes experimental results achieved by employinga library of probes according to the present invention to simultaneouslyscreen samples of cells for 54 different activated transcriptionfactors.

[0248]FIG. 6 provides the sequences for the probes used to form thetranscription factor probe library used in this experiment and theexperiments described in Sections 13-19 herein. It is noted that theprobes used were doubled stranded, FIG. 6 only showing the strand withbiotin labeled at the 5′ end. The strands not shown are the complementsto the sequences shown in FIG. 6.

[0249]FIG. 6 also shows the hybridization probes used in thehybridization probe array used in this experiment. FIG. 7 shows thelayout of the array of hybridization probes employed in the experimentsdescribed herein.

[0250] As can be seen in FIG. 7, each hybridization probe was placed inmultiple different regions, in this case, 4 separate regions. It isnoted that 2, 3, 4 or more multiple separate regions may be employed.Alternatively, only a single region may also be employed. As can also beseen in FIG. 7, the concentration of hybridization probe was varied inthe different regions. This serves both as an internal control as wellas a mechanism for quantifying the amount of immobilized probes.

[0251] Biotinylated oligonucleotides which are used as controls werepositioned in the regions of Row O and Column 17. These oligonucleotidesalso serve as a legend for the array, allowing a person to identifypositions of rows and columns in the array.

[0252] The transcription factor probe library described in FIG. 6 wascontacted with the array of hybridization probes described in regard toFIGS. 6 and 7. In this instance, unlike the experiments to be describedherein, no intermediate isolation of transcription factorprobe-transcription factor complexes was performed.

[0253]FIG. 8 is an image of the resulting array. As can be seen, all ofthe regions contain immobilized transcription factor probes. As can alsobe seen, the regions with higher concentrations of the samehybridization probe appear brighter (e.g., A1 and A2 vs. B1 and B2).

[0254] 13. Array Detection of Selected Activated Transcription Factors

[0255] Specific transcription factor probes and combinations oftranscription factor probes were also contacted with a nuclear extractfrom HeLa cells and probes from any transcription factorprobe-transcription factor complexes that formed being isolated. Thetranscription factor probes and combinations of transcription factorprobes used were Brn3, c-Myb, Smad3/4 individually and the combinationof Brn3, c-Myb, and Smad3/4.

[0256] FIGS. 9A-9D are images of the resulting arrays. As can be seen,only the hybridization probe regions for Brn3, c-Myb, and Smad3/4 appearto possess immobilized transcription factor probes in FIGS. 9A-9Crespectively. Meanwhile, FIG. 9D shows immobilized transcription factorprobes in the Brn3, c-Myb, and Smad3/4 hybridization probe regions. Theratios of the spot densities among Brn3, c-Myb, and Smad3/4 has beenfound to be very similar to what is observed in arrays with single probedetection.

[0257] As can also be seen, the higher concentration regions appearconsistently darker than the lower concentration regions. The use ofregions with different concentrations of hybridization probes allowsdifferent concentrations of transcription factor probes to be isolated.If the higher concentration region is saturated, the lower concentrationone can be used for evaluation.

[0258] 14. Array Detection of Activated Transcription Factors in HeLaCells

[0259] The entire library of probes described in regard to FIG. 6 wasalso contacted with a nuclear extract from HeLa cells and probes fromany transcription factor probe-transcription factor complexes thatformed being isolated.

[0260]FIG. 10B is an image of the resulting array. As a control, theentire library of probes described in regard to FIG. 6 was alsocontacted with a control sample that did not contain any transcriptionfactors. Probes from any transcription factor probe-transcription factorcomplexes that formed were isolated. Since no transcription factors arepresent in the control sample, it is expected that no complexes areformed and hence no probes are isolated. FIG. 10A is an image of theresulting array.

[0261] As can be seen in FIG. 10A and as would be expected, notranscription factor probes are immobilized in the array for the controlsample. Meanwhile, as can be seen in FIG. 10B, a myriad of transcriptionfactor probes are immobilized in the array for the HeLa cell sample.

[0262] 15. Comparison Between Activated Transcription Factors in HeLaCells and PMA-Activated HeLa Cells

[0263]FIG. 11A is an image of the array resulting from the entirelibrary of probes described in regard to FIG. 6 being contacted with anuclear extract from HeLa cells and any probes from any transcriptionfactor probe-transcription factor complexes that formed being isolated.

[0264] In comparison, FIG. 11B is an image of the array resulting fromthe entire library of probes described in regard to FIG. 6 beingcontacted with a nuclear extract from PMA-treated HeLa cells and probesfrom any transcription factor probe-transcription factor complexes thatformed being isolated.

[0265] As can be seen by comparing FIGS. 11A and 11B, a number oftranscription factors including Ets and NF-E1 can be seen to have beenactivated at higher levels in the PMA-treated HeLa cells.

[0266] The arrays shown in FIGS. 11A and 11B were imaged using aFluorChem imager (from Alpha Innotech Corp) in order to quantify theintensity of the spots appearing in the different regions of the array.FIGS. 12A and 12B provide tables of the signal intensity for the arraysshown in FIGS. 11A and 11B. FIG. 12C provides the ratio between theintensities shown in FIGS. 12A and 12B.

[0267] As can be seen by comparing the data shown in FIGS. 12A and 12B,the signal intensities for the regions associated with transcriptionfactors Egr (C5, C6, D5, D6); Ets (E9, E10, F9, F10); NF-E1 (G4, G5, H4,H5); and Smad3/4 (I15, I16, J15, J16) are more intense. Meanwhile, Ets(E9, E10, F9, F10) and Smad3/4 (I15, I16, J15, J16) show no differencein density.

[0268] The results of the experiment described in regard to FIGS. 11A,11B, and 12A-12C was confirmed by performing a standard gel shift assayusing Ets and NF-E1 probes. Specifically, nuclear extracts of HeLa andPMA-treated HeLa cells were incubated with Ets and NF-E1 probesrespectively. As can be seen in FIG. 13, bands corresponding to Etsappear for both PMA-treated and untreated HeLa cells. By contrast, aband corresponding to NF-E1 was not present in untreated HeLa cells butappeared in PMA-treated HeLa cells.

[0269] 16. Comparison Between Activated Transcription Factors in A431Cells and PMA-Activated A431 Cells

[0270] The entire library of probes described in regard to FIG. 6 wasalso contacted with a nuclear extract untreated A431 cells andPMA-treated A431 cells. FIG. 14A is an image of the array for untreatedA431 cells and FIG. 14B is an image of the array for PMA-treated A431cells. As can be seen by comparing FIGS. 14A and 14B, transcriptionfactors NF-E1 and NF-kB were found to be activated by PMA in A431 cells.

[0271] The results of the experiment described in regard to FIGS. 14Aand 14B was confirmed by performing a standard gel shift assay usingEts, NF-E1, and NF-kB probes. Specifically, nuclear extracts of A431 andPMA-treated A431 cells were incubated with Ets, NF-E1, and NF-kB probesrespectively. As can be seen in FIG. 15, bands corresponding to Etsappear for both PMA-treated and untreated A431 cells. By contrast, bandscorresponding to NF-E1 and NF-kB were not present in untreated A431cells but appeared in PMA-treated A431 cells.

[0272] 17. Comparison Between Activated Transcription Factors in JurkatCells and PMA-Activated Jurkat Cells

[0273] The entire library of probes described in regard to FIG. 6 wasalso contacted with a nuclear extract untreated Jurkat cells andPMA-treated Jurkat cells. FIG. 16A is an image of the array foruntreated Jurkat cells and FIG. 16B is an image of the array forPMA-treated Jurkat cells. As can be seen by comparing FIGS. 16A and 16B,transcription factor AP1 was found to be activated by PMA.Interestingly, NF-E1 was not induced by PMA, showing that NF-E1induction by PMA is cell line dependent.

[0274] 18. Comparison Of Activated Transcription Factors BetweenMultiple Cell Lines

[0275] The entire library of probes described in regard to FIG. 6 wasalso contacted with nuclear extracts for HeLa, A431, Jurkat, K-562, andY79 cells in order to compare the activated transcription factorspresent in these different cell lines. FIGS. 17A-17E show the resultingarrays for HeLa, A431, Jurkat, K-562, and Y79 cells respectively. As canbe seen, the mixture of transcription factors that are activated variesfor the different cells.

[0276] 19. Gel Shift Analyses Of Different Transcription Factors

[0277] Gel shift analyses were performed for multiple differenttranscription factors in order evaluate the sensitivity of gel shiftanalysis for detecting different transcription factors. Specifically, anuclear extract of HeLa cells was incubated with transcription factorprobes for c-Myb, Ets, Smad3/4, Brn3 and NF-E2. FIG. 18A is an image ofa polyacrylamide gel and FIG. 18B is an image of an agarose gel. As canbe seen in both figures, a band corresponding to probe-transcriptionfactor complexes is effectively separated. As can also be seen, c-Myb,Ets, and Smad3/4 can be detected. However, Brn3 and NF-E2 are difficultto detect. By contrast, c-Myb, Ets, Smad3/4, Brn3 and NF-E2 can all bedetected in the array shown in FIG. 10B.

[0278] It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compounds, compositions,kits, and methods of the present invention without departing from thespirit or scope of the invention. Thus, it is intended that the presentinvention cover the modifications and variations of this inventionprovided they come within the scope of the appended claims and theirequivalents.

1 162 1 21 DNA Artificial sequence Transcription factor probe PP01 1cgcttgatga ctcagccgga a 21 2 21 DNA Artificial sequence Transcriptionfactor probe PP02 2 ttccggctga gtcatcaagc g 21 3 26 DNA Artificialsequence Transcription factor probe PP03 3 gatcgaactg accgcccgcg gcccgt26 4 26 DNA Artificial sequence Transcription factor probe PP04 4acgggccgcg ggcggtcagt tcgatc 26 5 23 DNA Artificial sequenceTranscription factor probe PP05 5 gtctggtaca gggtgttctt ttt 23 6 23 DNAArtificial sequence Transcription factor probe PP06 6 aaaaagaacaccctgtacca gac 23 7 18 DNA Artificial sequence Transcription factorprobe PP07 7 cacagctcat taacgcgc 18 8 18 DNA Artificial sequenceTranscription factor probe PP08 8 gcgcgttaat gagctgtg 18 9 20 DNAArtificial sequence Transcription factor probe PP09 9 tgcagattgcgcaatctgca 20 10 20 DNA Artificial sequence Transcription factor probePP10 10 tgcagattgc gcaatctgca 20 11 27 DNA Artificial sequenceTranscription factor probe PP11 11 agaccgtacg tgattggtta atctctt 27 1227 DNA Artificial sequence Transcription factor probe PP12 12 aagagattaaccaatcacgt acggtct 27 13 27 DNA Artificial sequence Transcription factorprobe PP13 13 acccaatgat tattagccaa tttctga 27 14 27 DNA Artificialsequence Transcription factor probe PP14 14 tcagaaattg gctaataatcattgggt 27 15 25 DNA Artificial sequence Transcription factor probe PP1515 tacaggcata acggttccgt agtga 25 16 25 DNA Artificial sequenceTranscription factor probe PP16 16 tcactacgga accgttatgc ctgta 25 17 27DNA Artificial sequence Transcription factor probe PP17 17 agagattgcctgacgtcaga gagctag 27 18 27 DNA Artificial sequence Transcription factorprobe PP18 18 ctagctctct gacgtcaggc aatctct 27 19 25 DNA Artificialsequence Transcription factor probe PP19 19 atttaagttt cgcgcccttt ctcaa25 20 25 DNA Artificial sequence Transcription factor probe PP20 20ttgagaaagg gcgcgaaact taaat 25 21 27 DNA Artificial sequenceTranscription factor probe PP21 21 ggatccagcg ggggcgagcg ggggcca 27 2227 DNA Artificial sequence Transcription factor probe PP22 22 tggcccccgctcgcccccgc tggatcc 27 23 35 DNA Artificial sequence Transcription factorprobe PP23 23 gtccaaagtc aggtcacagt gacctgatca aagtt 35 24 35 DNAArtificial sequence Transcription factor probe PP24 24 aactttgatcaggtcactgt gacctgactt tggac 35 25 31 DNA Artificial sequenceTranscription factor probe PP25 25 ggaggagggc tgcttgagga agtataagaa t 3126 31 DNA Artificial sequence Transcription factor probe PP26 26attcttatac ttcctcaagc agccctcctc c 31 27 21 DNA Artificial sequenceTranscription factor probe PP27 27 gatctcgagc aggaagttcg a 21 28 21 DNAArtificial sequence Transcription factor probe PP28 28 tcgaacttcctgctcgagat c 21 29 21 DNA Artificial sequence Transcription factor probePP29 29 cggattgtgt attggctgta c 21 30 21 DNA Artificial sequenceTranscription factor probe PP30 30 gtacagccaa tacacaatcc g 21 31 32 DNAArtificial sequence Transcription factor probe PP31 31 cgaagtactttcagtttcat attactctac aa 32 32 32 DNA Artificial sequence Transcriptionfactor probe PP32 32 ttgtagagta atatgaaact gaaagtactt cg 32 33 27 DNAArtificial sequence Transcription factor probe PP33 33 cacttgataacagaaagtga taactct 27 34 27 DNA Artificial sequence Transcription factorprobe PP34 34 agagttatca ctttctgtta tcaagtg 27 35 41 DNA Artificialsequence Transcription factor probe PP35 35 gaccctagag gatctgtacaggatgttcta gatccaattc g 41 36 41 DNA Artificial sequence Transcriptionfactor probe PP36 36 cgaattggat ctagaacatc ctgtacagat cctctagggt c 41 3725 DNA Artificial sequence Transcription factor probe PP37 37 ctcagcttgtactttggtac aacta 25 38 25 DNA Artificial sequence Transcription factorprobe PP38 38 tagttgtacc aaagtacaag ctgag 25 39 22 DNA Artificialsequence Transcription factor probe PP39 39 ggaagcgaaa atgaaattga ct 2240 22 DNA Artificial sequence Transcription factor probe PP40 40agtcaatttc attttcgctt cc 22 41 25 DNA Artificial sequence Transcriptionfactor probe PP41 41 gatcccccca acacctgctg cctga 25 42 25 DNA Artificialsequence Transcription factor probe PP42 42 tcaggcagca ggtgttgggg ggatc25 43 25 DNA Artificial sequence Transcription factor probe PP43 43gatcgctcta aaaataaccc tgtcg 25 44 25 DNA Artificial sequenceTranscription factor probe PP44 44 cgacagggtt atttttagag cgatc 25 45 26DNA Artificial sequence Transcription factor probe PP45 45 ggaagcagaccacgtggtct gcttcc 26 46 26 DNA Artificial sequence Transcription factorprobe PP46 46 ggaagcagac cacgtggtct gcttcc 26 47 25 DNA Artificialsequence Transcription factor probe PP47 47 ttttggattg aagccaatat gataa25 48 25 DNA Artificial sequence Transcription factor probe PP48 48ttatcatatt ggcttcaatc caaaa 25 49 30 DNA Artificial sequenceTranscription factor probe PP49 49 acgcccaaag aggaaaattt gtttcataca 3050 30 DNA Artificial sequence Transcription factor probe PP50 50tgtatgaaac aaattttcct ctttgggcgt 30 51 27 DNA Artificial sequenceTranscription factor probe PP51 51 cgctccgcgg ccatcttggc ggctggt 27 5227 DNA Artificial sequence Transcription factor probe PP52 52 accagccgccaagatggccg cggagcg 27 53 27 DNA Artificial sequence Transcription factorprobe PP53 53 tggggaacct gtgctgagtc actggag 27 54 27 DNA Artificialsequence Transcription factor probe PP54 54 ctccagtgac tcagcacaggttcccca 27 55 22 DNA Artificial sequence Transcription factor probe PP5555 agttgagggg actttcccag gc 22 56 22 DNA Artificial sequenceTranscription factor probe PP56 56 gcctgggaaa gtcccctcaa ct 22 57 22 DNAArtificial sequence Transcription factor probe PP57 57 tgtcgaatgcaaatcactag aa 22 58 22 DNA Artificial sequence Transcription factorprobe PP58 58 ttctagtgat ttccattcga ca 22 59 27 DNA Artificial sequenceTranscription factor probe PP59 59 tacagaacat gtctaagcat gctgggg 27 6027 DNA Artificial sequence Transcription factor probe PP60 60 ccccagcatgcttagacatg ttctgta 27 61 27 DNA Artificial sequence Transcription factorprobe PP61 61 gaatggggca ctgaggcgtg accaccg 27 62 27 DNA Artificialsequence Transcription factor probe PP62 62 cggtggtcac gcctcagtgccccattc 27 63 26 DNA Artificial sequence Transcription factor probe PP6363 cgaattgatt gatgcactaa ttggag 26 64 26 DNA Artificial sequenceTranscription factor probe PP64 64 ctccaattag tgcatcaatc aattcg 26 65 28DNA Artificial sequence Transcription factor probe PP65 65 tgtcttcctgaatatgaata agaaataa 28 66 28 DNA Artificial sequence Transcriptionfactor probe PP66 66 ttatttctta ttcatattca ggaagaca 28 67 20 DNAArtificial sequence Transcription factor probe PP67 67 caaaactaggtcaaaggtca 20 68 20 DNA Artificial sequence Transcription factor probePP68 68 tgacctttga cctagttttg 20 69 27 DNA Artificial sequenceTranscription factor probe PP69 69 gatcctgtac aggatgttct agctaca 27 7027 DNA Artificial sequence Transcription factor probe PP70 70 tgtagctagaacatcctgta caggatc 27 71 31 DNA Artificial sequence Transcription factorprobe PP71 71 tcgagggtag ggttcaccga aagttcactc g 31 72 31 DNA Artificialsequence Transcription factor probe PP72 72 cgagtgaact ttcggtgaaccctaccctcg a 31 73 26 DNA Artificial sequence Transcription factor probePP73 73 agcttcaggt cagaggtcag agagct 26 74 26 DNA Artificial sequenceTranscription factor probe PP74 74 agctctctga cctctgacct gaagct 26 75 27DNA Artificial sequence Transcription factor probe PP75 75 gtgcatttcccgtaaatctt gtctaca 27 76 27 DNA Artificial sequence Transcription factorprobe PP76 76 tgtagacaag atttacggga aatgcac 27 77 16 DNA Artificialsequence Transcription factor probe PP77 77 agtatgtcta gactga 16 78 16DNA Artificial sequence Transcription factor probe PP78 78 tcagtctagacatact 16 79 39 DNA Artificial sequence Transcription factor probe PP7979 tcgagagcca gacaaaaagc cagacattta gccagacac 39 80 39 DNA Artificialsequence Transcription factor probe PP80 80 gtgtctggct aaatgtctggctttttgtct ggctctcga 39 81 21 DNA Artificial sequence Transcriptionfactor probe PP81 81 attcgatcgg ggcggggcga g 21 82 21 DNA Artificialsequence Transcription factor probe PP82 82 ctcgccccgc cccgatcgaa t 2183 22 DNA Artificial sequence Transcription factor probe PP83 83ggatgtccat attaggacat ct 22 84 22 DNA Artificial sequence Transcriptionfactor probe PP84 84 agatgtccta atatggacat cc 22 85 25 DNA Artificialsequence Transcription factor probe PP85 85 catgttatgc atattcctgt aagtg25 86 25 DNA Artificial sequence Transcription factor probe PP86 86cacttacagg aatatgcata acatg 25 87 24 DNA Artificial sequenceTranscription factor probe PP87 87 gatccttctg ggaattccta gatc 24 88 24DNA Artificial sequence Transcription factor probe PP88 88 gatctaggaattcccagaag gatc 24 89 33 DNA Artificial sequence Transcription factorprobe PP89 89 ctagagcctg atttccccga aatgatgagc tag 33 90 33 DNAArtificial sequence Transcription factor probe PP90 90 ctagctcatcatttcgggga aatcaggctc tag 33 91 21 DNA Artificial sequence Transcriptionfactor probe PP91 91 agatttctag gaattcaatc c 21 92 21 DNA Artificialsequence Transcription factor probe PP92 92 ggattgaatt cctagaaatc t 2193 20 DNA Artificial sequence Transcription factor probe PP93 93gtatttccca gaaaaggaac 20 94 20 DNA Artificial sequence Transcriptionfactor probe PP94 94 gttccttttc tgggaaatac 20 95 25 DNA Artificialsequence Transcription factor probe PP95 95 gcagagcata taaaatgagg tagga25 96 25 DNA Artificial sequence Transcription factor probe PP96 96tcctacctca ttttatatgc tctgc 25 97 32 DNA Artificial sequenceTranscription factor probe PP97 97 gatcgtaaga ttcaggtcat gacctgagga ga32 98 32 DNA Artificial sequence Transcription factor probe PP98 98tctcctcagg tcatgacctg aatcttacga tc 32 99 29 DNA Artificial sequenceTranscription factor probe PP99 99 agcttcaggt cacaggaggt cagagagct 29100 29 DNA Artificial sequence Transcription factor probe PP100 100agctctctga cctcctgtga cctgaagct 29 101 23 DNA Artificial sequenceTranscription factor probe PP101 101 cacccggtca cgtggcctac acc 23 102 23DNA Artificial sequence Transcription factor probe PP102 102 ggtgtaggccacgtgaccgg gtg 23 103 28 DNA Artificial sequence Transcription factorprobe PP103 103 agcttcaggt caaggaggtc agagagct 28 104 28 DNA Artificialsequence Transcription factor probe PP104 104 agctctctga cctccttgacctgaagct 28 105 15 DNA Artificial sequence Transcription factor probePP105 105 ctggaatttt ctaga 15 106 15 DNA Artificial sequenceTranscription factor probe PP106 106 tctagaaaat tccag 15 107 15 DNAArtificial sequence Transcription factor probe PP107 107 ctctgcgcccggccc 15 108 15 DNA Artificial sequence Transcription factor probe PP108108 gggccgggcg cagag 15 109 63 DNA Artificial sequence Hybridizationprobe MP02 109 ttccggctga gtcatcaagc gttccggctg agtcatcaag cgttccggctgagtcatcaa 60 gcg 63 110 78 DNA Artificial sequence Hybridization probeMP04 110 acgggccgcg ggcggtcagt tcgatcacgg gccgcgggcg gtcagttcgatcacgggccg 60 cgggcggtca gttcgatc 78 111 69 DNA Artificial sequenceHybridization probe MP06-1 111 aaaaagaaca ccctgtacca gacaaaaagaacaccctgta ccagacaaaa agaacaccct 60 gtaccagac 69 112 54 DNA Artificialsequence Hybridization probe MP08 112 gcgcgttaat gagctgtggc gcgttaatgagctgtggcgc gttaatgagc tgtg 54 113 60 DNA Artificial sequenceHybridization probe MP10 113 tgcagattgc gcaatctgca tgcagattgc gcaatctgcatgcagattgc gcaatctgca 60 114 81 DNA Artificial sequence Hybridizationprobe MP12 114 aagagattaa ccaatcacgt acggtctaag agattaacca atcacgtacggtctaagaga 60 ttaaccaatc acgtacggtc t 81 115 81 DNA Artificial sequenceHybridization probe MP14 115 tcagaaattg gctaataatc attgggttca gaaattggctaataatcatt gggttcagaa 60 attggctaat aatcattggg t 81 116 75 DNAArtificial sequence Hybridization probe MP16 116 tcactacgga accgttatgcctgtatcact acggaaccgt tatgcctgta tcactacgga 60 accgttatgc ctgta 75 11781 DNA Artificial sequence Hybridization probe MP18 117 ctagctctctgacgtcaggc aatctctcta gctctctgac gtcaggcaat ctctctagct 60 ctctgacgtcaggcaatctc t 81 118 75 DNA Artificial sequence Hybridization probe MP20118 ttgagaaagg gcgcgaaact taaatttgag aaagggcgcg aaacttaaat ttgagaaagg 60gcgcgaaact taaat 75 119 81 DNA Artificial sequence Hybridization probeMP22 119 tggcccccgc tcgcccccgc tggatcctgg cccccgctcg cccccgctggatcctggccc 60 ccgctcgccc ccgctggatc c 81 120 70 DNA Artificial sequenceHybridization probe MP24 120 aactttgatc aggtcactgt gacctgactt tggacaactttgatcaggtc actgtgacct 60 gactttggac 70 121 93 DNA Artificial sequenceHybridization probe MP26 121 attcttatac ttcctcaagc agccctcctc cattcttatacttcctcaag cagccctcct 60 ccattcttat acttcctcaa gcagccctcc tcc 93 122 63DNA Artificial sequence Hybridization probe MP28 122 tcgaacttcctgctcgagat ctcgaacttc ctgctcgaga tctcgaactt cctgctcgag 60 atc 63 123 63DNA Artificial sequence Hybridization probe MP30 123 gtacagccaatacacaatcc ggtacagcca atacacaatc cggtacagcc aatacacaat 60 ccg 63 124 96DNA Artificial sequence Hybridization probe MP32 124 ttgtagagtaatatgaaact gaaagtactt cgttgtagag taatatgaaa ctgaaagtac 60 ttcgttgtagagtaatatga aactgaaagt acttcg 96 125 81 DNA Artificial sequenceHybridization probe MP34 125 agagttatca ctttctgtta tcaagtgaga gttatcactttctgttatca agtgagagtt 60 atcactttct gttatcaagt g 81 126 82 DNAArtificial sequence Hybridization probe MP36 126 cgaattggat ctagaacatcctgtacagat cctctagggt ccgaattgga tctagaacat 60 cctgtacaga tcctctaggg tc82 127 75 DNA Artificial sequence Hybridization probe MP38 127tagttgtacc aaagtacaag ctgagtagtt gtaccaaagt acaagctgag tagttgtacc 60aaagtacaag ctgag 75 128 66 DNA Artificial sequence Hybridization probeMP40 128 agtcaatttc attttcgctt ccagtcaatt tcattttcgc ttccagtcaatttcattttc 60 gcttcc 66 129 75 DNA Artificial sequence Hybridizationprobe MP42 129 tcaggcagca ggtgttgggg ggatctcagg cagcaggtgt tggggggatctcaggcagca 60 ggtgttgggg ggatc 75 130 75 DNA Artificial sequenceHybridization probe MP44 130 cgacagggtt atttttagac cgatccgaca gggttatttttagaccgatc cgacagggtt 60 atttttagac cgatc 75 131 78 DNA Artificialsequence Hybridization probe MP46 131 ggaagcagac cacgtggtct gcttccggaagcagaccacg tggtctgctt ccggaagcag 60 accacgtggt ctgcttcc 78 132 75 DNAArtificial sequence Hybridization probe MP48 132 ttatcatatt ggcttcaatccaaaattatc atattggctt caatccaaaa ttatcatatt 60 ggcttcaatc caaaa 75 13390 DNA Artificial sequence Hybridization probe MP50 133 tgtatgaaacaaattttcct ctttgggcgt tgtatgaaac aaattttcct ctttgggcgt 60 tgtatgaaacaaattttcct ctttgggcgt 90 134 81 DNA Artificial sequence Hybridizationprobe MP52 134 accagccgcc aagatggccg cggagcgacc agccgccaag atggccgcggagcgaccagc 60 cgccaagatg gccgcggagc g 81 135 81 DNA Artificial sequenceHybridization probe MP54 135 ctccagtgac tcagcacagg ttccccactc cagtgactcagcacaggttc cccactccag 60 tgactcagca caggttcccc a 81 136 66 DNAArtificial sequence Hybridization probe MP56 136 gcctgggaaa gtcccctcaactgcctggga aagtcccctc aactgcctgg gaaagtcccc 60 tcaact 66 137 66 DNAArtificial sequence Hybridization probe MP58 137 ttctagtgat ttccattcgacattctagtg atttccattc gacattctag tgatttccat 60 tcgaca 66 138 81 DNAArtificial sequence Hybridization probe MP60 138 ccccagcatg cttagacatgttctgtaccc cagcatgctt agacatgttc tgtaccccag 60 catgcttaga catgttctgt a81 139 81 DNA Artificial sequence Hybridization probe MP62 139cggtggtcac gcctcagtgc cccattccgg tggtcacgcc tcagtgcccc attccggtgg 60tcacgcctca gtgccccatt c 81 140 78 DNA Artificial sequence Hybridizationprobe MP64 140 ctccaattag tgcatcaatc aattcgctcc aattagtgca tcaatcaattcgctccaatt 60 agtgcatcaa tcaattcg 78 141 84 DNA Artificial sequenceHybridization probe MP66 141 ttatttctta ttcatattca ggaagacatt atttcttattcatattcagg aagacattat 60 ttcttattca tattcaggaa gaca 84 142 60 DNAArtificial sequence Hybridization probe MP68 142 tgacctttga cctagttttgtgacctttga cctagttttg tgacctttga cctagttttg 60 143 81 DNA Artificialsequence Hybridization probe MP70 143 tgtagctaga acatcctgta caggatctgtagctagaaca tcctgtacag gatctgtagc 60 tagaacatcc tgtacaggat c 81 144 93DNA Artificial sequence Hybridization probe MP72 144 cgagtgaactttcggtgaac cctaccctcg acgagtgaac tttcggtgaa ccctaccctc 60 gacgagtgaactttcggtga accctaccct cga 93 145 78 DNA Artificial sequenceHybridization probe MP74 145 agctctctga cctctgacct gaagctagct ctctgacctctgacctgaag ctagctctct 60 gacctctgac ctgaagct 78 146 81 DNA Artificialsequence Hybridization probe MP76 146 tgtagacaag atttacggga aatgcactgtagacaagatt tacgggaaat gcactgtaga 60 caagatttac gggaaatgca c 81 147 64DNA Artificial sequence Hybridization probe MP78 147 tcagtctagacatacttcag tctagacata cttcagtcta gacatacttc agtctagaca 60 tact 64 148117 DNA Artificial sequence Hybridization probe MP80 148 gtgtctggctaaatgtctgg ctttttgtct ggctctcgag tgtctggcta aatgtctggc 60 tttttgtctggctctcgagt gtctggctaa atgtctggct ttttgtctgg ctctcga 117 149 63 DNAArtificial sequence Hybridization probe MP82 149 ctcgccccgc cccgatcgaatctcgccccg ccccgatcga atctcgcccc gccccgatcg 60 aat 63 150 66 DNAArtificial sequence Hybridization probe MP84 150 agatgtccta atatggacatccagatgtcc taatatggac atccagatgt cctaatatgg 60 acatcc 66 151 75 DNAArtificial sequence Hybridization probe MP86 151 cacttacagg aatatgcataacatgcactt acaggaatat gcataacatg cacttacagg 60 aatatgcata acatg 75 15272 DNA Artificial sequence Hybridization probe MP88 152 gatctaggaattcccagaag gatcgatcta ggaattccca gaaggatcga tctaggaatt 60 cccagaagga tc72 153 99 DNA Artificial sequence Hybridization probe MP90 153ctagctcatc atttcgggga aatcaggctc tagctagctc atcatttcgg ggaaatcagg 60ctctagctag ctcatcattt cggggaaatc aggctctag 99 154 63 DNA Artificialsequence Hybridization probe MP92 154 ggattgaatt cctagaaatc tggattgaattcctagaaat ctggattgaa ttcctagaaa 60 tct 63 155 60 DNA Artificialsequence Hybridization probe MP94 155 gttccttttc tgggaaatac gttccttttctgggaaatac gttccttttc tgggaaatac 60 156 75 DNA Artificial sequenceHybridization probe MP96 156 tcctacctca ttttatatgc tctgctccta cctcattttatatgctctgc tcctacctca 60 ttttatatgc tctgc 75 157 96 DNA Artificialsequence Hybridization probe MP98 157 tctcctcagg tcatgacctg aatcttacgatctctcctca ggtcatgacc tgaatcttac 60 gatctctcct caggtcatga cctgaatcttacgatc 96 158 87 DNA Artificial sequence Hybridization probe MP100 158agctctctga cctcctgtga cctgaagcta gctctctgac ctcctgtgac ctgaagctag 60ctctctgacc tcctgtgacc tgaagct 87 159 69 DNA Artificial sequenceHybridization probe MP102 159 ggtgtaggcc acgtgaccgg gtgggtgtaggccacgtgac cgggtgggtg taggccacgt 60 gaccgggtg 69 160 84 DNA Artificialsequence Hybridization probe MP104 160 agctctctga cctccttgac ctgaagctagctctctgacc tccttgacct gaagctagct 60 ctctgacctc cttgacctga agct 84 161 60DNA Artificial sequence Hybridization probe MP106 161 tctagaaaattccagtctag aaaattccag tctagaaaat tccagtctag aaaattccag 60 162 60 DNAArtificial sequence Hybridization probe MP108 162 gggccgggcg cagaggggccgggcgcagag gggccgggcg cagaggggcc gggcgcagag 60

1. A method for identifying which of a plurality of different activatedtranscription factors are present in a biological sample, the methodcomprising: contacting the biological sample with a library oftranscription factor (TF) probes under conditions where TF probe-TFcomplexes are formed between the TF probes in the library and activatedtranscription factors (TFs) present in the biological sample, the TFprobes each comprising a known recognition sequence varying within thelibrary, and the TF probes in the library being capable of binding to atleast two known, activated transcription factors; isolating the TFprobes that have bound to the activated TFs and formed the TF probe-TFcomplexes; and contacting the isolated TF probes with an array ofimmobilized hybridization probes under conditions suitable forhybridization of the strands of the TF probes to the hybridizationprobes in the array, wherein identification of the DNA probes bound tothe array identifies which of the plurality of different activated TFsare present in the biological sample.
 2. The method according to claim1, wherein each of the TF probes in the library is double-stranded DNA.3. The method according to claim 2, wherein one strand of the doublestranded TF probes further comprises a detectable marker, the methodfurther including using the detectable marker to identify which of theisolated TF probes hybridize to the array.
 4. The method according toclaim 2, wherein the detectable marker is at a 5′ end of the strand ofthe TF probes.
 5. The method according to claim 2, wherein thedetectable marker is biotin at a 5′ end of the strand of the TF probes.6. The method according to claim 1, wherein the library comprises TFprobes having recognition sequences between 10 and 100 base pairs inlength.
 7. The method according to claim 1, wherein the librarycomprises TF probes having recognition sequences between 20 and 40 basepairs in length.
 8. The method according to claim 1, wherein the librarycomprises TF probes having recognition sequences between 25 and 35 basepairs in length.
 9. The method according to claim 1, wherein the librarycomprises TF probes having at least 2 different recognition sequences.10. The method according to claim 1, wherein the library comprises TFprobes having at least 5 different recognition sequences.
 11. The methodaccording to claim 1, wherein the library comprises TF probes having atleast 10 different recognition sequences.
 12. The method according toclaim 1, wherein the library comprises TF probes having at least 20different recognition sequences.
 13. The method according to claim 1,wherein the library comprises TF probes having at least 50 differentrecognition sequences.
 14. The method according to claim 1, wherein therecognition sequences in the library of TF probes are for recognizingactivated transcription factors from at least 2 different types ofcells.
 15. The method according to claim 1, wherein the recognitionsequences in the library of TF probes are for recognizing activatedtranscription factors from at least 5 different types of cells.
 16. Themethod according to claim 1, wherein the recognition sequences in thelibrary of TF probes are for recognizing activated transcription factorsfrom at least 10 different types of cells.
 17. The method according toclaim 1, wherein the recognition sequences in the library of TF probesare for recognizing activated transcription factors from malignant,benign, and normal cell types.
 18. The method according to claim 1,wherein the biological sample is a nuclear extract of cells.
 19. Themethod according to claim 1, wherein each of the immobilizedhybridization probes comprises at least two copies of a complement to aportion of the recognition sequence comprised on TF probe.
 20. Themethod according to claim 1, wherein the recognition sequences comprisedon the TF probes are known to bind to at least two TFs selected from thegroup consisting of NF-E1, NFκB, Ets, Ap1, p53 and c-Myb.
 21. The methodaccording to claim 1, wherein the recognition sequences comprised on theTF probes are known to bind to at least two TFs selected from the groupconsisting of AP1, AP-2, ARE, Brn-3, C/EBP, CBF, CDP, c-Myb, CREB,E2F-1, EFR, ERE, Ets-1/PEA3, FAST-1, GAS/ISRE, GATA, GRE, HNF-4, IRF-1,MEF-1, MEF-2, Myc-Max, NF-1, NFATc, NF-E2, NFκB, Oct-1, p53, Pax-5,Pbx1, Pit 1, PPAR, PRE, RAR, RAR (DR-5), SIE, Smad SBE, Smad3/4, SP1,SRE, Stat1, Stat3, Stat4, Stat4, Stat5, Stat6, TFIIED, TR, TR(DR-4),USF-1, VDR (DR-3), HSE, and MRE;
 22. A method for identifying multipledifferent transcription factors present in a biological sample, themethod comprising: mixing the biological sample with a library oftranscription factor (TF) probes under suitable conditions such that theTF probes bind to the transcription factors (TFs) in the biologicalsample, each of the TF probes comprising a known recognition sequencevarying within the library, and the TF probes in the library beingcapable of binding to at least two known TFs; isolating the TF probesthat have bound to the transcription factors; and determining identitiesof isolated TF probes, determination of the identities identifying whichTFs are present in the biological sample.
 23. The method according toclaim 22, wherein the TF probes are double-stranded DNA.
 24. The methodaccording to claim 22, wherein the step of determining includes:sequencing the isolated TF probes, sequences of the TF probesidentifying which TFs are present in the biological sample.
 25. Themethod according to claim 22, wherein the step of determining includes:identifying the isolated TF probes by mass spectroscopy.
 26. The methodaccording to claim 22, wherein the step of determining includes:identifying the isolated TF probes by electrophoresis.
 27. The methodaccording to claim 22, wherein the library comprises TF probes havingrecognition sequences between 20 and 40 base pairs in length.
 28. Themethod according to claim 22, wherein the library comprises TF probeshaving at least 2 different recognition sequences.
 29. The methodaccording to claim 22, wherein the library comprises TF probes having atleast 5 different recognition sequences.
 30. The method according toclaim 22, wherein the library comprises TF probes having at least 10different recognition sequences.
 31. The method according to claim 22,wherein the recognition sequences in the library of TF probes are forrecognizing transcription factors from at least 2 different types ofcells.
 32. The method according to claim 22, wherein the recognitionsequences in the library of TF probes are for recognizing transcriptionfactors from at least 5 different types of cells.
 33. The methodaccording to claim 22, wherein the recognition sequences in the libraryof TF probes are for recognizing transcription factors from at least 10different types of cells.
 34. The method according to claim 22, whereinthe recognition sequences in the library of TF probes are forrecognizing TFs from malignant, benign, and normal cell types.
 35. Themethod according to claim 22, wherein the recognition sequencescomprised on the TF probes are known to bind to at least two TFsselected from the group consisting of AP1, AP-2, ARE, Brn-3, C/EBP, CBF,CDP, c-Myb, CREB, E2F-1, EFR, ERE, Ets, Ets-1/PEA3, FAST-1, GAS/ISRE,GATA, GRE, HNF-4, IRF-1, MEF-1, MEF-2, Myc-Max, NF-1, NFATc, NF-E1,NF-E2, NFκB, Oct-1, p53, Pax-5, Pbx1, Pit 1, PPAR, PRE, RAR, RAR (DR-5),SIE, Smad SBE, Smad3/4, SP1, SRE, Stat1, Stat3, Stat4, Stat4, Stat5,Stat6, TFIID, TR, TR(DR-4), USF-1, VDR (DR-3), HSE, and MRE.
 36. Themethod according to claim 22, wherein the biological sample is a nuclearextract of cells.